Journal of Environmental Science and Health Part B, 40:731–740, 2005Copyright C Taylor & Francis Inc. ISSN: 0360-1234 (Print); 1532-4109 (Online)DOI: 10.1080/03601230500189006
Comparison of Three Extraction Methods for 17β-Estradiol in Sand, Bentonite, and Organic-Rich Silt Loam
Soul Chun,1 Jaehoon Lee,1 Roland Geyer,2and David C. White2
1Biosystems Engineering and Soil Science Department, University ofTennessee, Knoxville, Tennessee, USA2Center for Biomarker Analysis, University of Tennessee, Knoxville, Tennessee, USA
Extraction is an important procedure for samples that contain soil because other com-pounds in soil may affect analysis of estrogens. This study was conducted to evaluatethree different extraction methods for 17β-estradiol in soil. Sand, bentonite, and organic-rich silt loam were spiked with 1 mg kg−1 of 17β-estradiol as a model compound of es-trogens. 17β-estradiol and its metabolites, estrone and estriol, were extracted using (i) amodified Bligh and Dyer extraction, (ii) a pressurized fluid extraction, and (iii) a diethylether extraction, and measured by liquid chromatography tandem mass spectrometry. There were significant differences in the extraction efficiency for 17β-estradiol amongthe extraction methods and the soils: the efficiencies ranged from 10% to 97%. Overall,the diethyl ether extraction method had the largest efficiency of 17β-estradiol with 45%and 57% for bentonite and silt loam, respectively. Transformation of 17β-estradiol toestrone and estriol in the different extraction methods was less than 3.6% during theextraction procedures. This study underlined the importance of sample preparation forestrogen analysis in soil samples. Key Words: 17β-estradiol; Estrone; Estriol; Extraction efficiency; Liquid chromatogra-phy; Tandem mass spectrometry.
Received October 22, 2004. Address correspondence to Soul Chun, Biological Resources and Technology Depart-ment, Yonsei University, 234 Meji-Ri, Hyongup-Meon, Wangwon-Do, Republic of Korea220-710; E-mail: firstname.lastname@example.org
Endocrine disrupting chemicals (EDCs) are defined as chemicals that can in-duce adverse health effects by disrupting an organism’s endocrine system ornormal development in vivo. The United States Geological Survey reportedthat 95 different organic wastewater contaminants, including EDCs (i.e., 17β-estradiol, estrone, and estriol), were detected in water from 139 streams in 30U.S. states during 1999 and 2000. Finlay-Moore, Hartel, and Cabrera alsoreported that concentrations of 17β-estradiol reached 150–2300 ng L−1 in therunoff from a field in which manure had been applied, indicating that a greatnumber of people and wildlife can be impacted by exposure to EDCs throughsoil-water systems. Although the human health risk associated with environ-mental exposure to EDCs is not clear, the compounds have been shown to induceestrogenic responses in fish at extremely low concentrations of less than 1 ngL−1.
With respect to analysis, the need for a sensitive, comprehensive, rapid, and
accurate detection method for estrogens has been emphasized in environmen-tal studies because contaminants are present at extremely low concentrationsand coexist with compounds that can interfere with the quantitative analysis,e.g., water-soluble organic materials and Ca2+ and Mg2+ ions.[5−7] Therefore,many sample preparation steps are required in order to concentrate samplesand to avoid interference with other compounds before analysis. At the samplepreparation stage, extracting estrogens from soil samples is difficult becauseestrogens have low water solubility (0.8–13.3 mg L−1) and these are moderatelyhydrophobic compounds (log Kow 2.6–4.0).[8−9] Estrogens are strongly sorbed tosoil and desorption is limited. In previous studies, Colucci, Bork, and Toppreported that less than 40% of 14C-17β-estradiol was extracted from soils after12 h of treatment. However, mineralization of 17β-estradiol in soils occurredslowly in their study (only 15% of 17β-estradiol was released as 14C-CO2 inthree months), and they concluded that the rest of 17β-estradiol remained insoils as a nonextractable form of 17β-estradiol or its metabolites.
The most common extraction methods for target contaminants in environ-
mental samples are solid-lipid extractions (i.e., Bligh and Dyer extractionand pressurized liquid extraction), solid-phase extraction, Soxhlet extraction,(ultra) sonication, microwave extraction, and supercritical fluid extraction. Insolid-lipid extractions, a solvent dissolves a target compound with a similarpolarity and moves it from a complex matrix to the solvent, or from the sol-vent to another solvent, during multistep extraction. This method was first re-ported by Folch et al. who used a solvent mixture of chloroform and methanoland then purified the extracts with aqueous KCl solution. Bligh and Dyermodified the Folch et al. method to improve extraction and purification ef-ficiencies for total lipid and develop a one-phase, initial, and rapid method. Subsequently, many modified Bligh and Dyer methods are currently used as
Three Extraction Methods for 17β-Estradiol
a standard solid-lipid extraction method.[13−14] Pressurized liquid extraction,however, has also been successfully applied to the extraction for soil, sludge,and other waste samples.[15−16] Casey et al. recently used a pressurized liquidextraction method to extract 17β-estradiol in soil.
The main objective of this study was to compare the commonly used solid-
lipid extraction methods—(i) a modified Bligh and Dyer extraction, (ii) a pres-surized liquid extraction, and (iii) a diethyl ether extraction—for the extractionefficiency of 17β-estradiol in soil. We hypothesized that diethyl ether would in-crease the extraction efficiency of estrogens because diethyl ether (log Kow 3.2)has a similar polarity of estrogens. In this study, 17β-estradiol was used as amodel compound of estrogens.
Sand (Rex International Co., Longview, TX) and pure Southern Bentonite(American Colloidal Co., Chicago, IL) were heated at 450◦C for 3 h to removeorganic matter. LaDelle silt loam (fine-silty, mixed, superactive, frigid, cumulicHapludoll) with 9.2% organic matter was dried and passed through a 2-mmsieve. To avoid microbial effects, all the soils were fumigated twice with chloro-form for 24 h in the dark and heated at 60◦C for 24 h to remove all chloroformvapors before starting experiments.
17β-estradiol, estrone, and estriol were purchased from Sigma-Aldrich (St.
Louis, MO). A stock solution of 5 mg L−1 17β-estradiol was made with methanol. The stock solution (200 µL) was spiked into 10 g of each soil sample to make aconcentration of 1 mg kg−1 of 17β-estradiol. The mass of 17β-estradiol, 10 µg, isequivalent to the mass of 17β-estradiol in approximately 6.7 g of hog manure.Two replicates were made. All glassware was washed with deionized water andoven-dried for 4 h at 450◦C to remove any organic contaminants.
Modified Bligh and Dyer Extraction Method
We used a single-phase chloroform and methanol buffer system designed
for the total lipid extraction method.[13−14] Ten mL of chloroform, 20 mL ofmethanol, and 8 mL of phosphate buffer (50 mM, pH 7.4) were added to a 10-gsoil sample and allowed to equilibrate for 3 h. Extraction of the single phase wascollected by centrifugation at 1000 g for 20 min and by decanting into anothertest tube. Five mL of chloroform was used to wash the pelleted solids, whichwere then vortexed for 5 min and recentrifuged. The supernatant was againdecanted and added to the first chloroform extract. An additional 5 mL of waterwas added to the extract and centrifuged at 1000 g for 20 min to separate theaqueous phase from the organic phases. The bottom layer for the organic phase,10 mL, was pipetted into a new test tube and dried under a stream of nitrogengas at 37◦C.
Samples for the pressurized fluid extraction method were prepared based
on a standard EPA method 3545 using Dionex Accelerated Solvent Extrac-tion 200 (Sunnyvale, CA). An 11-mL stainless steel extraction cell was used for10 g soil, and the solvent mixture acetone-hexane (1:1, v/v) was used for extrac-tion. Sample running conditions were selected as follows: oven temperaturewas 100◦C; system pressure was 1800 psi; static time was 5 min after a 5-minpreheat equilibration; flush volume was 60% of the cell volume, followed by a180-sec nitrogen purge at 150 psi. The extracted solution was dried under astream of nitrogen gas at 37◦C.
A 10-g soil sample was added to a test tube with 25 mL of diethyl ether.
The soil sample was vortexed for 5 min and centrifuged at 1000 g, 1500 rpm, for20 min. Aliquots of the organic layer, 10 mL, were collected and filtered throughglass wool packed in the bottom of a pipette. The extracted solution was driedunder a stream of nitrogen gas at 37◦C.
Analysis of 17β-Estradiol, Estrone, and Estriol
For the analysis of 17β-estradiol and its metabolites, all the extracted and
dried samples were redissolved in 1 mL of a 1:1 (v/v) mixture of methanoland a mobile phase (30% water: 70% acetonitrile, v/v). High-performance liq-uid chromatography (Hewlett-Packard 1100 system) coupled with electrosprayionization and tandem mass spectrometry (PE Sciex API 365, Concord, ON,Canada) was used to measure 17β-estradiol and its metabolites, i.e., estroneand estriol. Sample separation of estrogens was performed with a 30 × 4.1mm, 3 µm LUNA C18(2) column (Phenomenex Co., Torrance, CA). Flow rateof mobile phase was 50 µL min−1 with a gradient of 20–80% acetonitrile inwater containing 0.1% ammonium hydroxide (pH 10). The column separationtime was less than 2 min. A chromatogram of a standard mixture of estro-gens is shown in Figure 1. 17β-estradiol (C18H24O2), estrone (C18H22O2), andestriol (C18H24O3) were detected according to their molecular ions (Q1) at m/z271, m/z 269, and m/z 287, respectively, and characteristic fragment ions (Q3)at m/z 183, m/z 145, and m/z 171, respectively (Fig. 2). It was conducted onnegative electrospray ionization mode with the condition of −4200 V sprayvoltage and capillary temperature 425◦C. The limits of quantification based onthe ratio of signal to noise (S/N ≥ 10/1) for the compounds were 0.025, 0.010,and 0.025 mg L−1 for 17β-estradiol, estrone, and estriol, respectively. Good lin-earity of the standard calibration curves was obtained for concentrations from0.025 to 2.0 mg L−1 for 17β-estradiol (R2, 0.996), estrone (R2, 0.999), and estriol(R2, 0.999). Three Extraction Methods for 17β-Estradiol
Figure 1: The structures of 17β-estradiol, estrone, and estriol, and a chromatogram of astandard estrogen mixture. We used 200 µL of a 5 mg L−1 standard solution of estrogens.
Figure 3 shows the extraction efficiencies of three different extraction meth-ods for 17β-estradiol in soils. The extraction efficiencies ranged from 10% to97%. For sand, the diethyl ether extraction method (92%) was as good asthe pressurized fluid extraction method (97%) and was better than the mod-ified Bligh and Dyer extraction method (78%). The diethyl ether extractionmethod had the largest efficiency of 17β-estradiol in bentonite and silt loamwith 45% and 57%, respectively. The mass recovery in this study is consis-tent with previous studies that showed high sorption affinity.[10,17,20,21] In thestudy of Colucci, Bork, and Topp, the extractable 14C-17β-estradiol (1 mgkg−1) rapidly decreased, and the nonextractable 17β-estradiol in loam and siltloam was 70% and 56%, respectively, following three days of incubation. Theyalso reported that 17β-estradiol was transformed to estrone in a few hours andsuggested that analysis of estrogens in environmental samples should includeestrone.
One possible explanation that the diethyl ether extraction method had the
largest efficiency in Figure 3 involves the similar polarities of diethyl etherand estrogens. We believe that 17β-estradiol was more selectively extractedby diethyl ether than by other solvents. Again, estrogens are moderately hy-drophobic compounds and have a polarity similar to diethyl ether compared tothe solvents in the Bligh and Dyer extraction method and the pressurized fluidextraction method. However, the presence of water-soluble organic materialsin the environment, such as fulvic and humic acids, is one of the reasons whyestrogens can be overestimated when nonspecific methods of detection are usedto detect the compounds. Therefore, it is strongly recommended that analysisof estrogens in environmental samples should be based on a mass spectrometry
Figure 2: Product-ion-scan mass spectra of (a) 17β-estradiol, (b) estriol, and (c) estrone.
system, such as LC-MS/MS.[5,22] When nonspecific methods (e.g., spectroscopy)are used, diethyl ether can reduce interference with the hydrophilic water-soluble organic materials in soil and thus minimize their overestimation.
Table 1 shows the relative concentrations of estrone and estriol in this
study. The total of the metabolites in the three extraction methods was less than3.6% of the initial concentration of 17β-estradiol. Thus, transformation of 17β-estradiol to estrone or estriol did not significantly affect the extraction efficiencyof the three methods. The rest of the 17β-estradiol must have remained in thesoils and was not extracted by any of the three extraction methods. Aging isthought to be an explanation for the nonextractable 17β-estradiol in Table 1,and passive processes of aging include a number of intra-soil processes: sorptiononto soil particles, diffusion into spatially remote areas such as soil micropores,
-estr β β .04 .00 .01 .02 .05 .04 .02 .18 .03
-estr β † Relative × ND, § Total method)
Figure 3: Extraction efficiency (%) for 17β-estradiol from sand, bentonite, and LaDelle siltloam spiked with 1 mg kg−1 17β-estradiol.
and entrapment within soil organic matter. Casey et al. reported that thehigh sorption affinity of 17β-estradiol appeared to be associated with the surfacearea and/or the cation exchange capacity of soils. Further study including theaging effect will be necessary to establish a standard extraction method ofestrogens in soil samples.
This study shows the importance of sample preparation and extraction
methods for estrogen analysis in soil, although the spiked concentration of 17β-estradiol was higher than in real-world samples and, thus, our results may notbe representative of all soils. We also used disturbed soils (e.g., dried, sieved,heat-treated, autoclaved) in this study to mainly focus on comparing the sol-vent effect on extraction efficiency. However, we believe that this study providesimportant preliminary data with respect to the analysis of estrogens in soil andwater systems.
There is a great need to develop and evaluate a standardized analysis methodfor estrogens in soil/water samples in order to systematically determine thefate and transport of estrogens. We evaluated three different methods for theirextraction efficiencies for 17β-estradiol in soil samples. The three methodsshowed significantly different extraction efficiencies in various soils, implyingthe importance of extraction procedures at the sample preparation stage for
Three Extraction Methods for 17β-Estradiol
quantitative analysis of estrogens in environmental study. Overall, we showedthe possibility of increasing the efficiency of estrogens in soil samples withdiethyl ether extraction.
We would like to acknowledge the National Science Foundation award no. 0244169 for supporting this research.
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