Scientific paper
Determination of Estrogens in Water Samples Using Dispersive Liquid Liquid Microextraction and High Performance Liquid Chromatography
Mohammad Reza Hadjmohammadi* and Seyedeh Saboora Ghoreishi
Department of chemistry, University of Mazandaran, Babolsar, 47416-95447, Iran
* Corresponding author: E-mail: hadjmr@umz.ac.ir Tel & fax: +981125342350
Received: 24-12-2010
Abstract
A new method for the analysis of estrogens including estrone (E1), 17P- estradiol (E2) and diethylstilbestrol (DES) in aqueous samples was performed using dispersive liquid liquid microextraction (DLLME) and high- performance liquid chromatography (HPLC). In order to optimize DLLME some important parameters such as type and volume of extraction and disperser solvent, extraction time, ionic strength and pH of sample were studied and optimum condition was obtained. Under optimum condition (extraction solvent: 80 |L CCl4; dispersive solvent: 1.25 mL acetone; NaCl: 12% (w/w) and pH of sample = 10.0), the enrichment factors and extraction recoveries were 71.0-78.5 and 85.2-94.2 respectively. Linearity was observed in the range of 0.02-500.0 |g L-1 for DES and 0.03-500.0 |g L-1 for E1 and E2. Limits of detection were 0.008 |g L-1 for DES and 0.010 |g L-1 for E1 and E2. The relative standard deviations (RSDs) for determination of estrogens in water were in the range of 2.4-3.2% (n = 5).
Keywords: Dispersive liquid liquid microextraction, Estrogens, Water samples, HPLC.
1. Introduction
The endocrine- disrupting phenomenon became a relatively new area of concern. Endocrine- disrupting compounds (EDCs) are environmental contaminants that disturb normal endocrine function.1'2 Among the wide range of substances with endocrine- disrupting properties, natural and synthetic estrogens are of particular interest due to their high estrogenic potency.34 Estrogens mimic such as alkylphenols, nonylphenols, bisphenol A and alkylphenol polyethoxylates.5-9 The major estrogenic components that have been identified as the natural estrogens, include estrone (E1) and 170-estradiol (E2) which are either produced endogenously by animals or used as pharmaceutical products in both human and veterinary medicine.1011 One of the synthetic forms of the estrogen hormone, diethylstilbe-strol (DES) is used as a growth promoter in domestic ani-mal.12 More than 30 years of research have confirmed that DES is a teratogen, an agent that can cause malformations of an embryo. It is reported that exposure to synthetic estrogen during critical stages of child development in the uterns increases the risk of abnormalities which can result in struc-
tural, functional or long term pathological changes including cancer. E1, E2 and DES have shown estrogenic effects in fish at very low concentration.1314 Thus the very low environmental concentrations expected for these estrogens require a sensitive, selective and simple method to monitor them in water.15 Before determination of these materials in water samples they require a pretreatment technique. Many different pretreatment techniques, such as liquid liquid extraction (LLE),16-18 solid phase extraction (SPE),819 20 solid phase microextraction (SPME),21-23 stir bar sorptive extraction (SBSE),24 cloud point extraction (CPE)25 and liquid phase microextraction techniques26-30 were used for the extraction of estrogens. Unfortunately, the traditional methods such as LLE and SPE require a large consumption of organic solvents, sample volume and are time consuming. Although SPME and SBSE are both solvent-free techniques, but the fibers of SPME are fragile, expensive and have limited life time and sample carry over is the other problem of this technique. For SBSE an additional desorption step is required when it couples with HPLC. CPE uses surfactants for extraction thus the choices of the surfactants often bring the nuisance to the analysis of analytes using GC and
HPLC.31-35 Dispersive liquid liquid microextraction (DLLME) is a novel microextraction method which was developed by Assadi and co-workers.36 It is based on a ternary component solvent system and uses microliter volumes of extraction solvent. In this method, the appropriate mixture of extraction solvent and disperser solvent is injected into aqueous sample by syringe, rapidly. Thereby, cloudy solution is formed. It has been widely used for trace analysis in different matrices (water, serum and urine etc).37-39 DLLME is a rapid, simple and low cost method with high recovery and enrichment factor.40-43 The aim of this work was to use DLLME-HPLC-UV for simultaneous determination of three estrogens in water samples. The effects of various experimental parameters on the extraction of estrogens from water samples were studied and optimized. The optimized method was applied to determine estrogens in river tap and well water.
2. Experimental
2. 1. Materials and Solutions
Estrone (99.0%), 170-estradiol (98.0%) and diethyl-stilbestrol (99.0%) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Chloroform (analytical grade), carbon tetrachloride (spectrophotometric grade) and ace-tonitrile (HPLC-grade) were purchased from Fluka (Buchs, Switzerland). Dichloromethane (analytical grade), acetone and methanol (HPLC-grade), sodium hydroxide, hydrochloric acid, sodium chloride and tetrahydro-furan (THF) were obtained from Merck (Darmstadt, Germany). Water used was double distilled deionized. Stock solutions of estrogens (500.0 mg L-1) were prepared in methanol and stored in the dark at 4 °C. The working solutions were prepared daily by an appropriate dilution of the stock solution in water. All solutions were filtered through 0.45 pm membrane filters (Millipore, Bedford, MA) prior to use.
bottom and spiked at level of 10.0 pg L-1 of target analy-tes. 1.25 mL of acetone (as disperser solvent) containing 80 pL CCl4 (as extraction solvent) were rapidly injected into sample solution and the mixture was gently shaken. In this step, a cloudy solution was formed and the analytes in the water sample were extracted into fine droplets. The mixture was centrifuged for 10 min at 4000 rpm and CCl4 was sedimented in the bottom of the conical test tube (about 60 pL). The sedimented phase was removed by mi-crosyringe, placed into a vial and evaporated by slight heating to dryness. The residue was reconstituted by 50pL methanol and 20 pL of this solution was injected into HPLC.
2. 4. Calculation of Enrichment Factor and Extraction Recovery
Enrichment factor (EF) was defined as the ratio of the analyte concentration in the sedimented phase (Csed) to the initial concentration of analyte (C0) within the sample:
gp _ Cjçd Co
(1)
Csed for each estrogen compound was obtained from calibration curves of standard solutions. The extraction recovery (ER) and it's relationship with EF are as the following.
ER = Csed Vs«d x ] QQ
CoVo
ER = EFx^ixl00
(2)
(3)
Vo
Where Vsed and V0 are the volumes of sedimented phase and aqueous phase, respectively.
2. 2. Instrumentation
Chromatographic measurements were carried out using a HPLC system equipped with a series 10 LC pump, UV detector model LC-95 set at 245 nm and model 7125i manual injector with a 20 pL sample loop (Perkin-Elmer, Norwalk, CT, USA). Adjustment of pH of solutions was done by a 3030 Jenway pH meter (Leeds, UK). Column used was C18 (250 x 4mm, 5pm particle size) from waters (Milford, MA, USA). Mobile phase used was a mixture of acetonitrile / water /THF (50:48:2 v/v) at a flow rate of 1.0 mL min-1 and room temperature.
2. 3. Extraction Procedure
For the DLLME, 5.0 mL of aqueous sample was placed in a 10 mL screw cap glass test tube with conical
3. Results and Discussion
There are various parameters affecting the DLLME performance, including type and volume of extraction and disperser solvent, ionic strength, extraction time and pH of sample. These parameters were investigated and the optimal condition was selected.
3. 1. Optimization of DLLME
3. 1. 1. Selection of Extraction and Dispersive Solvents
The selection of an appropriate extraction and dispersive solvents are very important for the DLLME process. Some of significant parameters in selection of ex-
traction solvent are (a) higher density than water (b) good chromatographic behavior (c) extraction capability of interested compounds and (d) low solubility in water. In addition, dispersive solvent should be miscible with both water and the extraction solvent. In this study, all combinations of extraction solvents (dichloromethane: CH2Cl2, chloroform: CHCl3 and carbon tetrachloride: CCl4) and disperser solvents (methanol, acetonitrile and acetone,) were tested. The experimental procedure was done by injecting each combinations of 1.0 mL of dispersive solvent containing 50 pL of extraction solvent into 5.0 mL water sample. In the case of CH2Cl2, as extraction solvent, a two-phase system was not observed with any studied dispersive solvents. In the case of CHCl3 just acetone and acetonitrile were formed two-phase system but were not stable. With CCl4 as extraction solvent, a stable two-phase system was formed with all three dispersive solvents. These cloudy solutions centrifuged and sedimented phases were injected to HPLC. Among these dispersive solvents, acetone gave the highest recovery with CCl4. Thereby, CCl4 and acetone were selected as the best extraction and dispersive solvent, respectively. Results are shown in Fig.1.
constant and 80 pL of CCl4 was chosen as the optimum of extraction volume.
Figure 1. Effect of different dispersive solvent on the extraction recovery of estrogens (n = 3). Extraction condition: sample volume, 5.0 mL; dispersive solvent volume (acetone, acetonitrile and methanol), 1.0 mL; extraction solvent, 50 pL CCl4.
3. 1. 2. Effect of Extraction Solvent Volume
In order to study the effect of extraction solvent volume, a series of experiment were performed using 1.0 mL acetone containing different volumes of CCl4 (20-120 pL). Fig. 2 shows the ER versus volume of extraction solvent. It is obvious that ER gradually increases by increasing the volume of extraction solvent up to 80 pL of CCl4 because the capability of sample extraction into organic phase is increased. After this volume the ER was almost
Figure 2. Effect of the volume of extraction solvent (CCl4) on the extraction recovery of estrogens (n = 3). Dispersive solvent: 1.0 mL of acetone. Other conditions as Fig. 1.
3. 1. 3. Effect of Dispersive Solvent Volume
Volume of the dispersive solvent is one of the important factors which must be considered in DLLME. At low volume, acetone can not disperse extraction solvent properly, and cloudy solution does not form completely, and the extraction recoveries are low. On the contrary, the solubility of analytes in water sample increases at high volu-
Figure 3. Effect of the volume of dispersive solvent (acetone) on the extraction recovery of estrogens (n = 3). Extraction solvent (CCl4) volume: 80 pL. Other conditions as Fig. 2.
me of acetone, therefore, extraction recovery decreases too. In this work various volumes of acetone were tested (0.25-1.75 mL) with optimum amount of CCl4 (80 pL) and results are shown in Fig. 3. It was observed that the extraction recovery was increased by increasing the volumes of acetone up to 1.25 mL and then decreased. Thus, 1.25 mL of acetone was chosen as the optimum volume.
3. 1. 4. Effect of Ionic Strength
Generally, increasing the ionic strength of sample solution decreases the solubility of the analyte and enhances extraction recovery. To investigate the influence of ionic strength on the extraction recovery, various experiments were performed by adding different amounts of Na-Cl 0.0-18.0% (w/w). Fig. 4 shows the effect of the ionic strength on the extraction recovery of estrogens. It was found that addition of NaCl up to 12.0% (w/w) causes to increase extraction recovery due to salting out effect and moving of analytes into the organic droplets. At higher concentration of NaCl (higher than 12.0%) interaction between salt ions and analytes reduce the ability of analy-te to move into organic droplets and decrease the extraction recovery. Therefore 12.0% (w/w) of NaCl was selected as the optimum of ionic strength.
recovery. It is revealed that after formation of the cloudy solution, the surface area between extraction solvent and water sample is infinitely large and shows that the transition of analytes from water sample to extraction solvent is fast and equilibrium state is achieved quickly. Thus, cen-trifugation procedure could be done immediately after forming cloudy solution. Preliminary consideration of centrifugation time in the range 2 to 15 minutes showed that 10 minutes centrifugation leads to better aggregation of sedimented phase at bottom of the conical test tube and provided the highest recovery values for estrogens. So, in this method the most time-consuming step is the centrifu-ging of sample solution in the extraction procedure, which is 10 min.
3. 1. 6. Effect of Sample pH
To examine the effect of sample pH on extraction recovery, hydrochloric acid and sodium hydroxide were used to adjust acidity (pH = 5.0-7.0) and basicity (pH = 8.0-12.0), respectively. Fig. 6 shows the curve of recoveries of estrogens versus sample pH (5.0-12.0). Results show that extraction recoveries increased with increasing pH value in the range of 5.0 to 10.0, and then decreased at higher pH values (because ionized forms of these compounds at pH higher than 10). Thus pH = 10.0 was selected as the optimum of sample pH.
120 -
Figure 5. Effect of pH on the extraction recovery of estrogens (n = 3). NaCl: 12% (w/w).Other conditions as Fig. 4.
NaCl (w/w %)
Figure 4. Effect of the ionic strength (addition of NaCl to sample solution) on the extraction recovery (n = 3). Extraction conditions: sample volume, 5.0 mL; extraction solvent (CCl4): 80 |L; dispersive solvent (acetone): 1.25 mL.
3. 1. 5. Effect of Extraction Time
In DLLME, extraction time is defined as interval time between injecting the mixture of disperser solvent (acetone) containing of extraction solvent (CCl4) and before starting to centrifuge. The effect of extraction time was examined in the range of 0-60 min. Results showed that extraction time has little influence on the extraction
3. 2. Optimization of Chromatographic Conditions for Separation of Estrogens
In order to select a suitable mobile phase composition for separation of estrogens, several preliminary experiments were carried out. Methanol and acetonitrile (ACN) were examined as organic modifiers where ACN provide better resolution and peak shapes. It is observed
that variation of mobile phase pH had no significant effect on resolution also addition of a small amount of THF to the mobile phase reduced the tailing of estrogens peak without loss of resolution between them. Investigation of different volume percentage of acetonitrile (45-60% v/v), THF (1.0-3.0% v/v) on resolution between analytes peaks and matrix peaks showed that the appropriate mobile phase composition for the separation of estrogens was a mixture of acetonitrile / water /THF (50:48:2 v/v). At this condition estrogen peaks in the chromatograms had no interferences with other compounds extracted from water sample. Chromatogram of spiked water samples with standard of estrogens were shown in Fig. 6 and 7.
3. 3. Figures of Merit
Under the optimum condition, limits of detection (LODs), limits of quantification (LOQs), linear range (LR), correlation coefficient (r), intra-day precision (repeatability) and inter-day precision (reproducibility), extraction recovery and enrichment factor of the DLLME method for the estrogens were obtained and shown in Table 1. LOD and LOQ was determined based on 3Sb/m and 10Sb/m, respectively, where Sb is the standard deviation of blank and is equal to Peak-Peak noise when only mobile phase was passing through the column for 45 min and »m« is the slope of calibration curve. Limits of detection were 0.008 pg L-1 for DES and 0.010 pg L-1 for E1 and E2. Linear range was in the range of 0.020-500.0 pg L-1 for DES and 0.030-500.0 pg L-1 for E1 and E2. Correlation coefficient (r) ranged from 0.9997 to 0.9998. Relative
Table 1. Quantitative results of estrogens determined by DLLME-HPLC-UV from water samples a.
Parameters
E1
Compounds E2
DES
Limit of detection	0.010	0.010	0.008 (LOD, |g L -1)
Limit of quantification 0.030	0.030	0.020 (LOQ, |g L -1)
Linear range	0.030-500.0 0.030-500.0 0.020-500 (LR, |g L-1)
Correlation coefficient (r) 0.9997	0.9997	0.9998
Intra-day precision	2.4	3.2	2.7 (RSD %, n = 5)
Inter-day precision	4.9	3.5	4.1 (RSD %, n = 5)
Extraction recovery (ER %) 85.2	94.5	88.4
Enrichment factor (EF) 71.0	78.5	73.5
a Extraction conditions: water sample volume, 5.0 mL 20 |g L-1 of each estrogen; extraction solvent (CCl4) volume, 80 |L; disperser solvent (acetone) volume, 1.25 mL; NaCl: 12% (w/w); pH = 10.0. HPLC conditions: Mobile phase: water/acetonitrile/THF (50:48:2 v/v); flow rate: 1.0 mL min-1; column: C18 (250 X 4 mm, 5|im); room temperature; X = 245 nm.
standard deviations (RSD) were used to determine the in-tra-day precision and inter-day precision of the method. In this way, consecutive extraction of five aqueous samples spiked at 20 pg L-1 (working standard solution) was performed in a day and five continual days to evaluate the in-tra-day and inter-day precision of the estrogens recovery. Results are shown in Table 1.
3. 4. Real Water Analysis
River and well water from (Babolsar and Babol city, Iran) and tap water from our laboratory were collected and extracted using the optimized DLLME method. The extracts were analyzed by HPLC-UV. The results for well and tap water showed that any of the estrogens were detected above the detection limits of the method. In Babol-sar and Babol river water samples E1, E2 and DES were detected and they were confirmed by spiking with standards of estrogens. Concentration of analytes in both river waters was determined using standard addition method. Recoveries and concentration of estrogens in Babolsar and Babol river, well and tap water are shown in Table 2. Figs. 6 and 7 show the chromatograms obtained from Ba-bol and Babolsar river, respectively.
Table 2. Determination of estrogens and their recoveries in water samples a.
Com-	Sample	Concentration	Recovery
pounds		(Pg L-1)	(%) c
	Mean ± SD n = 5 b		Mean ± SD n = 5b
E1	Babolsar river	7.3 ± 0.4	84.7 ± 6.4
	Bobol river	2.7 ± 0.2	83.6 ± 5.4
	Well water	NDd	86.9 ± 3.9
	Tap water	ND	90.5 ± 6.8
E2	Babolsar river	9.7 ± 0.3	93.4 ± 3.1
	Bobol river	2.2 ± 0.2	92.5 ± 5.8
	Well water	ND	91.8 ± 4.6
	Tap water	ND	89.9 ± 6.1
DES	Babolsar river	6.1 ± 0.2	89.6 ± 5.1
	Bobol river	4.3 ± 0.1	88.3 ± 5.4
	Well water	ND	88.2 ± 2.5
	Tap water	ND	95.8 ± 7.3
a Extraction and HPLC conditions: as Table 1. b n: Number of replication
c Spiked at 10 |g L-1 d Non detected
4. Conclusions
Determination of estrogens in tap, river and well water samples were performed using DLLME-HPLC-UV. DLLME provide a simple, inexpensive, repeatable and easy to use method for extraction and preconcentration of
Table 3. Comparison of DLLME-LC-UV with other methods for the determination of estrogens.
Method	Compound	Recovery (%)	LOD	LR	Extraction	
			(TO L-1)	L-1)	Time (min,)	References
SPE-LC-UV	E1	90-102	0.0162	0.1-400	NA a	[19]
	E2	85-105	0.0781	0.1-300	NA	
	DES	86-112	0.00098	0.01-300	NA	
SBSE-LC-UV	E1	24.6	1.0	0.005-0.050	120	[24]
	E2	11.1	1.0	0.005-0.050	120	
CPE-LC-UV	E1	98	0.25	1-192	60	[25]
	E2	81.2-92	0.32	1-90	60	
HF-LPME-LC-UV b	DES	87.1-92.6	0.5	1-100	40	[26]
DLLME-SFO-LC-UV c	E1	89-100	1.0-1.6	5-1000	0	[29]
	E2	102-116	0.8-1.4	5-1000	0	
DLLME-LC-UV	E1	84.7-90.5	0.01	0.03-500.0	0 d	Present
	E2	89.9-94.5	0.01	0.03-500.0	0	study
	DES	88.2-95.8	0.008	0.02-500.0	0	
a Non available b Hollow fiber-mediated liquid phase microextraction c Dispersive liquid-liquid microextraction with solidification of a floating organic drop d See text for extraction time definition
estrogens in environmental water samples. In comparison with other method for extraction and determination of estrogens the presented method has high recovery, low limit of detection and good linearity with a short extraction time as shown in Table 3.
Figure 6. Chromatograms of estrogens at optimum extraction condition a) Babol river water b) spiked Babol river water (10 |g L-1). Mobile phase: acetonitrile /water /THF (50:48:2 v/v); flow rate: 1.0 mL min-1; column: C18 (250 X 4 mm, 5|im); room temperature; X = 245 nm.
Figure 7. Chromatograms of estrogens at optimum extraction condition a) Babolsar river water b) spiked Babolsar river water (10 |g L-1). Other Condition as Fig. 6.
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Povzetek
Izdelana je bila nova metoda za določevanje estrogenov (estrona- E1, 17 P - estradiola- E2 in dietilstilbestrola -DES) v vodnih vzorcih, ki temelji na disperzijski mikroekstrakciji tekoče-tekoče (DLLME) in visokoločljivostni tekočinski kro-matografiji (HPLC). Pogoji ekstrakcije so bili določeni na osnovi izbire topil, njihovega volumna, ekstrakcijskega časa, ionske moči in pH vrednosti. Pri optimalnih pogojih (ekstrakcijsko topilo 80 |L CCl4, disperzijsko topilo 1,25 mL acetona, 12 % raztopina NaCl in pH 10) so bili doseženi koncentracijski faktorji od 71do 78,5 in izkoristki ekstrakcije med 85,2 in 94,2 %. Linearnost metode je bila v območju od 0,02 do 500 |g L-1 za DES in od 0.03 do 500.0 |g L-1 za E1 in E2. Dosežene meje zaznave so bile 0.008 |g L-1 za DES in 0.010 |g L-1 zaE1 in E2. Relativni standardni odmik pri določevanju estrogenov v vodnih vzorcih je bil od 2.4 do 3.2 % (n = 5).