Research On The Determination Of Sex Hormones By High Performance Liquid Chromatography

Sex hormones can maintain the stability of the endocrine system, thus, it is very significant to monitor the hormone levels of mammalian in clinical disease prevention, diagnosis and treatment. At present, sex hormones are widely used in the production of animal husbandry. However, owing to illegal utilize of the sex hormones, there are many hormone residues in animal products and environmental pollutants. Therefore, it is quite necessary to build some accurate and convenient methods for the determination of sex hormones to protect people's health and keep the ecological balance. In this paper, we have studied several methods based on high performance liquid chromatography to determine1713-estradiol (BE2), estrone (El), diethylstilbestrol (DES) and progesterone (PES). The main research contents are as follows:Part1Ultrasound-assisted surfactant-enhanced emulsification microextraction (UASEME) combined with HPLC for the determination of sex hormonesChapter1UASEME-HPLC for the determination of sex hormones in water samplesThe UASEME method was adopted to extract BE2, El and DES in environmental water samples. The optimal condition was2.4x10-6mol/L Triton X-100as the dispersant and emulsifier,50μL CCl4as the extractant. Then, the mixed solution was placed in an ultrasonic water bath for ultrasonication at35kHz and25℃for5min to form a uniform emulsion. The emulsion was then detached by centrifugation for5min at4000rpm. After phase separation, the enrichment factor of the analytes was over85.29. Via global optimization, this method was characterized by an acceptable linear range of10to1000ng/mL for BE2, El and DES, a validated accuracy and precision (RSD:0.85-1.28%), with LOD of0.10-0.20ng/mL and good recoveries (=89.82%). This method shortened theextraction time and improved the extraction efficiency. Chapter2UASEME-HPLC for the determination of sex hormones in blood, urine and milk samplesThe UASEME method was adopted to extract sex hormones in biological and milk samples. The protein precipitants of1mLACN and0.3g MgSO4,0.2g Na2SO4,0.1g NaAc,0.4g MgSO4and0.5mL ACN were used for the whole blood, human urine and milk, respectively. Then,0.8x10-5mol/L Triton X-100as the dispersant and emulifier,80 pL CCI4as the extractant were added in the solution which was diluted to pH=7.0. The mixed solution was kept in an ultrasonic water bath for ultrasonication at35kHz and25℃for3min. The emulsion was then detached by adding0.3g NaCl and centrifugation for5min at4000rpm. Under the optimum conditions, this method was characterized by an acceptable linear range of5to1000ng/mL for BE2, El and DES,10to1000ng/mL for PES, a validated accuracy and precision (RSD:1.28-3.88%), with LOD of0.05-0.50ng/mL, and high recoveries (=83.2%). The method was successfully applied to analyze sex hormones in blood, urine and milk samples, with accurate, convenient and potential application.Part2Determination of sex hormones in human urine sample by HPLC with ultrasound assisted cloud point extraction (UA-CPE)The UA-CPE method was developed to analysis BE2, El and DES in human urine samples, combined with HPLC.0.5%(v/v) Tergitol TMN-6was adopted as the extractant. The proper extraction condition was ultrasonication at35kHz and45℃for45min. Then, the solution was detached by2.0%NaCl and centrifugation for5min at4000rpm. The enrichment factor was higher than78.39. The good linear range of BE2, El and DES was from5.0to1000ng/mL. The relative standard deviation over3days (RSD) was0.84-5.15%, and the LOD was0.10-0.20ng/mL, with the good recoveries (=85.35%). The method shortened the extraction time and improved the extraction efficiency and stability. Part3Determination of sex hormones in milk samples by HPLC with salting-out assisted liquid-liquid extraction (SA-LLE)The SA-LLE was adopted ACN,NaAc and MgSO4to extract sex hormnes in milk. The optimal experiment conditions:6mL acidical ACN,0.6g NaAc and2.4g MgSO4was added into15mL milk, centrification twice at10000rpm for3min. The good linear was observed in the range of10-5000ng/mL for E2, Eland DES, and20-8000ng/mL for PES. The limit of detection ranged in0.40-2.40ng/mL, and the relative standard deviations were0.3-4.4%(RSD), with good recoveries over95.1%which was better than liquid-liquid extraction and extraction after protein precipation. The method can be feasiblely applied to the analysis of sex hormones residues in milk samples.