Analysis Of Hormone Residues In Aquatic Products By Subcritical Fluid Extraction-high Performance Liquid Chromatography System

Subcritical fluid extraction is a new extraction technology, which can achievebetter extraction efficiency under relatively low pressure. And it can address theshortcomings of supercritical CO₂extraction, so subcritical fluid extraction has drawnmore and more attention. Due to its commercial availability, the permanent dipolemoment （2.05D） and reasonable critical properties （101.1°C,4.06MPa） of R134ahave led to the evaluation of its use as an alternative to supercriticalCO₂for theextraction of polar compounds. Hormonal chemicals can promote the anabolism ofprotein so as to quicken animal’s growth and improve the conversion rate of feed.They have been widely used in stockbreeding, which significantly improve theproductivity of husbandry, but the residue in animal food could endanger the health ofconsumers. However, because of profit motive, some of anabolic hormones are stillsecretly used in some farms. Therefore, it is essential to establish a reliable, fast,convenient and green analytical method to control the illegal use of steroids andmonitor its contents in fishery products. The aim of this paper is to develop asubcritical R134a extraction-high performance liquid chromatography method for thedetermination of various hormone residues in aquatic samples.Orthogonal array design and Response surface methodology （RSM） wereadopted to optimize the parameters of testosterone propionate,17α-methyltestosteroneand medroxyprogesterone extraction with subcritical R134a. The extracts werepurified with liquid-liquid extraction and C₁₈-solid phase extraction （SPE）, and thenquantified by HPLC. Base on the analysis of the results, the effects of the extractionpressure, extraction temperature, extraction time, and co-solvent volume wereexplored. The extraction conditions of three sex hormones were optimized differently,and the results showed in the following:1. Orthogonal array design was designed for extraction of testosteronepropionate with subcritical R134a fluid; the methanol solution collected was purifiedwith petroleum ether, and then quantified by HPLC. It was predicted that the optimumextraction conditions within the experimental ranges would be the pressure14MPa,temperature23℃, dynamic extraction time40min, and co-solvent volume2mL. Thedescending impact of the three factors for the extraction efficiency of testosterone propionate was extraction pressure, extraction time and extraction temperature. Thismethod has high sensitivity and fine reproducibiliy. The limit of quantification was2.0μg/kg. Mean recovery of testosterone propionate was above90%, RSD valueswere at less than5%.2. RSM was designed for extraction of17α-methyltestosterone with subcriticalR134a fluid; the methanol solution collected was purified with C18-solid phaseextraction （SPE）, and then quantified by HPLC. It was predicted that the optimumextraction conditions within the experimental ranges would be the pressure5MPa,temperature31℃, and co-solvent volume3.35mL. This method has high sensitivityand fine reproducibiliy. The limit of quantification was5μg/kg. Mean recovery of17α-methyltestosterone was above90%, RSD values were at less than3%. Thevariable with the largest effect on the extraction rate was the linear term of co-solventvolume), followed by the linear terms of pressure and temperature. The quadraticterms of pressure and co-solvent volume, as well as the interactions between pressureand co-solvent volume, had significant effects on the extraction rate.3. RSM was designed for extraction of medroxyprogesterone with subcritical R134afluid; the methanol solution collected was purified with C18-solid phase extraction（SPE）, and then quantified by HPLC. It was predicted that the optimum extractionconditions within the experimental ranges would be the pressure3MPa, temperature25℃, and co-solvent volume6mL. This method has high sensitivity and finereproducibiliy. The limit of quantification was5μg/kg. Mean recovery ofmedroxyprogesterone was above95%, RSD values were at less than5%. The variablewith the largest effect on the extraction rate was the linear term of co-solvent volume,followed by the quadratic terms of co-solvent volume. The interactions betweenpressure and co-solvent volume, as well as the interactions between temperature andco-solvent volume, had significant effects on the extraction rate.It is for the first time at home and abroad that subcritical R134a extraction wasused as pretreatment technology for residue analysis. Rather than supercritical CO₂extraction, subcritical R134a extraction has low extraction pressure and temperature.So it can be applied in large-scale application. In addition, this method takes aboutonly1h, the organic solvent used was only1/10compared with conventional method,and the analysis result is accurate and reliable. Besides, the steps of extraction andseparation can be finished at the same time, the operating cycle is short, and themethod is less environmental pollution.