RP-HPLC法测定鸡胚蛋及鸡蛋中性激素的残留量

目的:建立了RP-HPLC法测定鸡胚蛋及鸡蛋中己烯雌酚、丙酸睾酮激素残留.方法:采用Waters XTerra C18柱(150mm×4.6mm,5μm),流动相为甲醇:乙腈:水(2:2:1,V/V/V),流速为1ml/min,柱温为40℃,检测波长为240hm.用外标法定量,测定激素残留量.结果与结论:该方法线性范围为0.05～3.500μg/ml,标准曲线线性关系良好,其回归方程和相关系数分别为:己烯雌酚Y=21734x 963.18,R2=0.999l;丙酸睾酮Y=28967x-475.62,R2=0.9995.最低检测限为己烯雌酚0.25ng,丙酸睾酮0.33ng.回收率在78.7%～98.4%之间.相对标准偏差小于2.1%.     

液相色谱-串联质谱法测定动物肌肉和牛奶中的雌二醇

目的 建立丹磺酰氯衍生液相色谱-串联四极杆质谱检测鸡肉、牛肉、牛奶中雌二醇的高灵敏分析方法。方法酶解后的样品用HLB和氨基固相萃取柱富集净化,氮气吹干后,在60 ℃条件下丹磺酰氯衍生5 min;以乙腈-水（含0.1%乙酸）为流动相,采用多离子反应监测(MRM)正离子模式进行检测。结果 鸡肉、牛肉、牛奶中雌二醇的检出限为0.6 ng/kg,定量限为2.0 ng/kg,在3个添加水平的平均回收率在82.0%~88.9%之间,相对标准偏差均小于7.3%。市售牛肉、鸡肉和牛奶中17-β-雌二醇含量的中位数（平均值）分别为5.51 ng/kg (66.78 ng/kg)、4.60 ng/kg (16.94 ng/kg)和18.00 ng/kg (16.80 ng/kg),九十百分位数（P90）分别为248.91 ng/kg、39.33 ng.kg和39.60 ng/kg 结论 本方法适用动物肌肉和乳中超痕量雌二醇的含量检测。牛肉、鸡肉和牛奶中17-β-雌二醇的大样本监测数据可为科学的制定残留限量提供必要的数据基础。     

超高效液相色谱-串联质谱法测定水产品及其制品中3种抗生素和14种性激素残留量

目的建立超高效液相色谱-串联质谱法(ultra performance liquid chromatography-tandem mass spectrometry,UPLC-MS/MS)测定水产品及其制品中3种抗生素和14种性激素残留量的分析方法。方法样品经乙酸乙酯提取,GCB/NH2固相萃取小柱净化,洗脱液氮气吹至近干,用甲醇-水(1:9,V:V)溶液溶解,采用大气压电离离子(atmospheric pressure chemical ionization,APCI)源,正负离子模式采集,多反应监测方式(multiple reaction monitoring, MRM)检测,基质匹配外标法,定性和定量分析氯霉素、甲砜霉素、氟甲砜霉素、丙酸睾酮、睾丸酮、甲基睾丸酮、雌酮、雌二醇、雌三醇、己烯雌酚、醋酸甲地孕酮、炔诺酮、醋酸氯地孕酮、诺龙、康力龙、苯丙酸诺龙、群勃龙的残留量。结果 17种激素在2～200μg/L范围内线性兲系良好,相兲系数均大于0.99,定量限为1.5～3μg/kg;添加水平为5～15μg/kg,平均回收率在65%～100%,相对标准偏差为1%～9%。结论该方法简单、快捷,可作为水产品及其制品中17种激素的检测方法。     

钼蓝比色法测定保健食品中微量硒

建立了钼蓝比色法测定保健食品中微量硒的新方法。该方法最大吸收波长760nm,在0～100ng/mL浓度范围内浓度与吸光度呈线性关系,回归方程y=-4.142＋133.2x,相关系数r=0.9991,最低检测量1ng/mL,重复性好,RSD=3.17%,回收率95.98%～105.00%。该方法简便、快速、灵敏、准确,适合不同补硒类保健食品中的硒含量测定,该方法的建立有利于这类产品质量控制和提高。     

基于黑磷纳米片的荧光适配体传感器检测雌激素17β-雌二醇

建立一种定量检测雌激素17β-雌二醇（E2）的荧光适配体传感器。以黑磷晶体为原料,利用超声辅助液相剥离技术制备了黑磷纳米片（BPNs）作为荧光受体,基于6-羧基荧光素标记的适配体（FAM-Apt）与BPNs间的荧光共振能量转移机制构建了荧光适配体传感器,对BPNs浓度、FAM-Apt浓度和反应时间进行优化,并对自来水和牛奶样品进行测定。结果表明,该BPNs具有独立的片层结构且粒径均匀;在最优条件下,即BPNs和FAM-Apt的浓度分别为10 μg/mL和7.5 nmol/L时,该传感器对E2检测的线性范围为1.5～60 ng/mL,检测限为1.0 ng/mL。自来水和牛奶样品中加标回收率分别为91.2%～104.8%和87.5%～104.3%;相对标准偏差（RSD）为4.99%～10.53%和4.48%～11.24%。该方法简便、灵敏,30 min即可完成检测,特异性强,能够实现对实际样品中E2的定量检测。     

奶粉中雌二醇的高效液相色谱法检测

建立高效液相色谱法检测奶粉中雌二醇的方法。优化实验条件,采用紫外检测器,检测波长为230 nm。方法对奶粉中雌二醇的检出限为5 ng/g。在低、中、高3个加标水平下,奶粉中雌二醇的回收率在81.0%～91.9%的范围内,相对标准偏差在3.71%～4.74%范围内(n=4)。该法可有效监控奶粉中雌二醇的残留量。     

保健食品中黄酮含量三种测定方法研究

对比色法、紫外分光光度法、高效液相色谱法对保健食品中黄酮含量的测定进行了系统研究,其线性范围、回归方程、相关性、最低检测量、精确度、回收率均达到满意的效果,可作为定量测定依据.其中比色法在10～60 μg/mL,(y=4.667×104-7.8×10-3x,r=0.9995),紫外分光光度法在5～25μg/mL(y=1.420×10-3+0.028x,r=0.9998),高效液相色谱法在10～60μg/mL(y=3.3776×104+4.03×104x,r=0.9989)范围内具有良好的线性;3种方法的回收率分别为:98.6%～108.4%,98.0%～101.2%,95.4%～99.5%:RSD为0.68%,0.24%,1.24%;最低检测量为1 μg/mL,0.5μg/mL,2 ng/mL.这些方法的建立不仅为该产品质量控制提供有效保证,也为同类产品中黄酮含量测定提供有益借鉴.     

HPLC法同时测定油茶籽油中的γ-生育酚、β-谷甾醇和角鲨烯

本研究建立了一种高效液相色谱法同时测定油茶籽油中γ-生育酚、β-谷甾醇和角鲨烯。油茶籽油样品经过皂化、萃取、浓缩,将油茶籽油中的皂化物类成分除去,富集其中的不皂化物类成分,并通过HPLC进行分析检测,按外标法测定其含量。HPLC色谱条件:色谱柱Hypersil ODS C18(250 mm×4.6 mm,5μm);流动相为100%甲醇,等度洗脱;流速0.7 mL/min;柱温30℃;检测波长:205 nm。结果表明γ-生育酚、β-谷甾醇和角鲨烯分别在0.01~0.40μg(R~2=0.9999)、0.44~10.94μg(R~2=1)、0.01~0.55μg(R~2=0.9990)范围内呈较好的线性关系,相关系数均接近1。平均加样回收率分别为97.1322%(RSD=1.28%)、100.2346%(RSD=1.54%)、98.0859%(RSD=1.58%),相对标准偏差均小于3%。结论:该方法前处理简单易行,操作简便,灵敏度高,重现性和准确性好,可用于油茶籽油中γ-生育酚、β-谷甾醇和角鲨烯的含量测定和油茶籽油质量控制。     

用于检测牛奶中雌二醇的酶热传感技术研究

目的建立一种用于检测牛奶中雌二醇的酶热传感技术。方法羧基化雌二醇经1-乙基-(3-二甲基氨基丙基)碳酰二亚胺盐酸盐(EDC)与β-内酰胺酶偶联。酶热传感器中装SPG酶柱,通过ELISA原理使游离雌二醇与结合雌二醇竞争结合雌二醇抗体,从而检测基质中的雌二醇浓度。优化酶热传感检测体系中的反应底物、系统流速、抗原抗体稀释比例等实验因素。结果磷酸盐缓冲液(PBS)基质中测定IC50值为10.2 ng/m L,线性检测范围:5.1~19.4 ng/m L,最低检测限2.8 ng/m L,变异系数为3.4%,与多种雌激素类结构类似物之间均无交叉反应,特异性良好。牛奶样品中测定IC50值:0.45 ng/m L,线性检测范围:0.24~0.79 ng/m L,最低检测限0.12ng/m L。结论酶热传感技术可以快速检测牛奶中的雌二醇残留,操作便捷、样品前处理简单,是一种高效、经济的适用于食品安全工作现场检测的方法。     

同位素稀释高压液相色谱-串联质谱法测定鱼虾中丙酸睾酮残留量

目的采用高压液相色谱-串联质谱(high pressure liquid chromatography-tandem mass spectrometry,HPLC-MS/MS)快速检测鱼虾等水产品中丙酸睾酮残留。方法向捣碎匀浆后的样品中加入内标诺龙-D3混匀后,叔丁基甲醚超声提取,-80℃冷冻30 min后12000 r/min离心净化。以Eclipse Plus C18为色谱分离柱,以乙腈-0.1%甲酸水溶液为流动相,流速为0.3 mL/min,采用三重四极杆质谱在正离子模式下进行选择反应离子监测。结果丙酸睾酮在0.5~100 ng/mL范围内呈线性关系,相关系数为0.9989;方法定量限为0.5μg/kg。当添加水平为0.5~10.0μg/kg时,平均回收率为71.2%~104.5%,相对标准偏差为2.26%~5.65%。结论该方法操作简单、快速、灵敏度高,适用于快速检测水产品中丙酸睾酮的残留量。     

Fate of endogenous steroid hormones in steer feedlots under simulated rainfall-induced runoff

Steroid hormones pose potential risks to fish and other aquatic organisms at extremely low concentrations. To assess the factors affecting the release of endogenous estrogenic and androgenic steroids from feedlots during rainfall, runoff, and soil samples were collected after simulated rainfall on a 14-steer feedlot under different rainfall rates and aging periods and analyzed for six steroid hormones. While only 17α-estradiol, testosterone, and progesterone were detected in fresh manure, 17β-estradiol, estrone, and androstenedione were present in the surficial soil after two weeks. In the feedlot surficial soil, concentrations of 17α-estradiol decreased by approximately 25% accompanied by an equivalent increase in estrone and 17β-estradiol. Aging of the feedlot soils for an additional 7 days had no effect on estrogen and testosterone concentrations, but androstenedione concentrations decreased substantially, and progesterone concentrations increased. Androstenedione and progesterone concentrations in the surficial soil were much higher than could be accounted for by excretion or conversion from testosterone, suggesting that other potential precursors, such as sterols, were converted after excretion. The concentration of androgens and progesterone in the soil were approximately 85% lower after simulated rainfall, but the estrogen concentrations remained approximately constant. The decreased masses could not be accounted for by runoff, suggesting the possibility of rapid microbial transformation upon wetting. All six steroids in the runoff, with the exception of 17β-estradiol, were detected in both the filtered and particle-associated phases at concentrations well above thresholds for biological responses. Runoff from the aged plots contained less 17α-estradiol and testosterone, but more estrone, androstenedione, and progesterone relative to the runoff from the unaged plots, and most of the steroids had a lower particle-associated fraction.     

Analysis of Infant Formula for Steroid Hormones by Gas Chromatography–Tandem Mass Spectrometry Using Microwave-Assisted Extraction and Gel Permeation Chromatography Clean Up

A new method was developed for effective separation and simultaneous determination of estrogens, gestagens, and androgens, including estrone, 17β-estradiol, testosterone, progesterone, and estriol, in infant formula. The samples were enzymatically digested with β-glucuronidase/arylsulfatase prior to microwave-assisted extraction. After the extract was cleaned up by gel permeation chromatography the hormones were derivatived with 50 μL BSTFA containing 1 % TMCS. The derivatived hormones were measured with GC–MS/MS using electron impact ionization source in the positive multi-reaction monitoring mode. The calibration curves showed good linearity with correlation coefficient (r) of >0.999. The limit of quantification of five hormones ranged from 0.094 to 0.265 μg kg^?1, which is below the minimum required performance limits established by the European Community. The intra- and inter-day precision (as RSD) for six determinations of five analytes at 40 μg kg^?1 spiked level was in range of 3.4–5.4 % and 3.5–6.8 %, respectively. The recovery of five analytes was obtained to be 84.5–104 %, with relative standard deviations (RSD, n?=?6) of 1.7–5.5 %. This method has been successfully used for the qualitatively and quantitatively determination of estrone, 17β-estradiol, testosterone, progesterone, and estriol in infant formula.     

Natural occurrence of steroid hormones in food

The natural occurrence of the sex steroid hormones progesterone, testosterone, 17β-estradiol and estrone in food was investigated in a survey of the German market basket. The main metabolic precursors, intermediates and metabolites (pregnenolone, androstenedione, hydroxyprogesterone, dehydroepiandrosterone (DHEA), dihydrotestosterone, androsterone, 17α-estradiol and estriol) were also included in the investigation. Particular attention was paid to DHEA, which is said to have anti-aging properties. Analysis was carried out by gas chromato-graphy-mass spectrometry (GC-MS). The steroid patterns of pork, meat products, fish and poultry resemble those known for beef. Milk and milk products reflect the hormone profile of female cattle with high amounts of progesterone, which accumulates with increasing milk fat content. Milk products supply about 60–80% of ingested female sex steroids. Eggs are a considerable source of any of the investigated steroids and contribute to the nutritional hormone intake in the same order as meat and fish (10–20%). In vegetable food no estrogens could be detected. Plants supply testosterone in the same order as meat and milk products (20–40%) though. They contain considerable amounts of hormone precursors as well (contribution to DHEA supply: about 80%). In comparison to the human daily production of steroid hormones the nutritional supply (about 10 μgd⁻¹ progesterone, 0.05 μg d⁻¹ testosterone, 0.1 μg d⁻¹ estrogens, 0.5 μg d⁻¹ DHEA) is insignificant.     

量子点荧光免疫检测牛奶中己烯雌酚、雌二醇

研究牛奶中己烯雌酚、雌二醇的荧光免疫检测技术并对实际样品进行检测。辅助量子点标记技术,结合间接竞争酶联免疫吸附法原理建立检测标准曲线,评价特异性,对实际样品进行检测。己烯雌酚的荧光免疫检测在0.472 ng/mL~66.597 ng/mL内呈线性关系,最低检测限为0.236 ng/mL。雌二醇在0.418 ng/mL~195.065 ng/mL内呈线性关系,最低检测限为0.109 ng/mL。建立一种快速检测牛奶中己烯雌酚的荧光免疫检测方法,方法可同时运用于雌二醇的检测,特异性与回收率良好,可补充传统方法用于实际样品中雌激素类残留测定。     

Anaerobic transformation kinetics and mechanism of steroid estrogenic hormones in dairy lagoon water

Wastewater from concentrated animal feeding operations (CAFOs) frequently contains high concentrations of steroid estrogenic hormones. Release of these hormones into the environment may occur when CAFO wastewater is applied to agricultural lands as a nutrient and water source for crop production. To assess the potential risk of hormone contaminants derived from animal wastewater, we investigated the transformation kinetics and mechanisms of three natural estrogenic hormones (17α-estradiol, 17β-estradiol, and estrone) in aqueous solutions blended with dairy lagoon water under anaerobic conditions. Initial transformations of the three hormones in the dairy lagoon water were dominated by biodegradation and the degradation rates were temperature-dependent. The total amounts of hormones (initial concentration at 5 mg L(-1)) remaining in the solution after 52 days at 35 °C accounted for approximately 85%, 78%, and 77% of the initial amounts of 17α-estradiol, 17β-estradiol, and estrone, respectively. This observation suggests that these hormones are relatively stable over time and may accumulate in anaerobic or anoxic environments and anaerobic CAFO lagoons. A racemization reaction between 17α-estradiol and 17β-estradiol via estrone was observed in aqueous solutions in the presence of CAFO wastewater under anaerobic conditions. The initial hormone concentrations did not affect this degradation mechanism. A reversible reaction kinetic model was applied to fit the observed transformation dynamics. The degradation and regeneration of the parent hormone and its metabolites were successfully simulated by this model. The information in this study is useful for assessing the environmental risk of steroid hormones released from CAFO wastewater and to better understand why these hormone contaminants persist in many aquatic environments.     

Hormone discharges from a midwest tile-drained agroecosystem receiving animal wastes

Manure is increasingly being viewed as a threat to aquatic ecosystems due to the introduction of natural and synthetic hormones from land application to agricultural fields. In the Midwestern United States, where most agricultural fields are tile-drained, there is little known about hormone release from fields receiving animal wastes. To this end, seven sampling stations (four in subsurface tile drains and three in the receiving ditch network) were installed at a Midwest farm where various types of animal wastes (beef, dairy, and poultry lagoon effluent, dairy solids, and subsurface injection of swine manure) are applied to agricultural fields. Water flow was continuously monitored and samples were collected for hormone analysis during storm events and baseline flow for a 15 month study period. The compounds analyzed included the natural hormones 17α- and 17β-estradiol, estrone, estriol, testosterone, and androstenedione and the synthetic androgens 17α- and 17β-trenbolone and trendione. Hormones were detected in at least 64% of the samples collected at each station, with estrone being detected the most frequently and estriol the least. Testosterone and androstendione were detected more frequently than synthetic androgens, which were detected in fewer than 15% of samples. Hormone concentrations in subsurface tile drains increased during effluent irrigation and storm events. Hormones also appeared to persist over the winter, with increased concentrations coinciding with early thaws and snowmelt from fields amended with manure solids. The highest concentration of synthetic androgens (168 ng/L) observed coincided with a snowmelt. The highest concentrations of hormones in the ditch waters (87 ng/L for total estrogens and 52 ng/L for natural androgens) were observed in June, which coincides with the early life stage development period of many aquatic species in the Midwest.     

Differential effects of testosterone and 17β-estradiol on gonadal development in five anuran species

Sex hormones are essential for sexual differentiation and play a key role in the development of gonads in amphibians. The goal of this study was to evaluate the influence of exogenous sex steroids, testosterone, and 17β-estradiol (E(2)) on development of gonads in five anuran species differing in their evolutionary positions, sex determination, and mode of gonadogenesis. We found that in two closely related species of fire-bellied toad, Bombina bombina and Bombina variegata, testosterone and E(2) exposure results in sex reversal as well as intersex and undifferentiated gonads. Similarly, sex reversal was observed in Hyla arborea after exposure to male or female sex steroids. Xenopus laevis was sensitive to E(2) but only moderately to testosterone. In Bufo viridis, treatment with either sex hormone provoked a developmental delay in gonads and Bidder's organs. Therefore, susceptibility to hormonal sex reversal appeared species dependent but unrelated to genetic sex determination and the type of gonadogenesis. We also found that the onset of sex steroid exposure influences gonad differentiation and the meiotic status of the germ cells depends on their location within the gonad. Our findings reveal differential sensitivity of amphibians to testosterone and E(2), establishing a hierarchy of sensitivity to these hormones among different anuran species.     

Occurrence of hormonally active compounds in food: a review

 The present review gives an overview of the occurrence of hormones, hormone mimics, and hormone antagonists in food. The first part deals comprehensively with concentrations of the human sex steroid hormones progesterone, 17β-estradiol estrone, and testosterone in animal and vegetable food. The dietary intake of steroid hormones (10 μg/day progesterone, 0.1 μg/day estrogens, and 0.05 μg/day testosterone) is negligible compared to the human endogenous hormone synthesis. The second part addresses the phytoestrogens (isoflavones, coumestans, other bioflavonoids, lignans, phytosterols), which occur in food in much higher amounts than steroid hormones. Therefore, they can cause hormonal effects although their estrogen equivalents (relative to 17β-estradiol) are estimated to be 10^–2–10^–4. These effects can be beneficial or adverse, depending on the effectiveness and amount of the ingested hormone agonist, synergistic, and antagonistic effects with other dietary or endogenous hormones, interactions with other dietary compounds (e.g. fiber and fat intake), and the hormonal status of the individual. The review also summarizes the occurrence of steroid hormone precursors and of other growth-related hormones in food (corticosteroids, indole-3-carbinol, protein hormones). It ends with the presentation of residues and contaminants of fungal or anthropogenic origin (mycoestrogens, pesticides, plastic or food additives, industrial chemicals) which have also shown hormonal or hormone-blocking properties. Received: 12 August 1998 / Revised version: 3 November 1998