A new dispersive liquid–liquid microextraction using ionic liquid based microemulsion coupled with cloud point extraction for determination of copper in serum and water samples

Ecotoxicology and Environmental Safety 126 (2016) 186–192

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A new dispersive liquid–liquid microextraction using ionic liquid based microemulsion coupled with cloud point extraction for determination of copper in serum and water samples Salma Aslam Arain a,n, Tasneem Gul Kazi a, Hassan Imran Afridi a, Mariam Shahzadi Arain a, Abdul Haleem Panhwar a, Naeemullah Khan a, Jameel Ahmed Baig a, Faheem Shah b a b

National Centre of Excellence in Analytical Chemistry, University of Sindh, Jamshoro 76080, Pakistan Department of Chemistry, COMSATS Institute of Information Technology, Abbottabad 22060, Pakistan

art ic l e i nf o

a b s t r a c t

Article history: Received 22 August 2015 Received in revised form 26 December 2015 Accepted 28 December 2015

A simple and rapid dispersive liquid–liquid microextraction procedure based on ionic liquid assisted microemulsion (IL-mE-DLLME) combined with cloud point extraction has been developed for preconcentration copper (Cu2 þ ) in drinking water and serum samples of adolescent female hepatitits C (HCV) patients. In this method a ternary system was developed to form microemulsion (mE) by phase inversion method (PIM), using ionic liquid, 1-butyl-3-methylimidazolium hexafluorophosphate ([C4mim][PF6]) and nonionic surfactant, TX-100 (as a stabilizer in aqueous media). The Ionic liquid microemulsion (IL-mE) was evaluated through visual assessment, optical light microscope and spectrophotometrically. The Cu2 þ in real water and aqueous acid digested serum samples were complexed with 8-hydroxyquinoline (oxine) and extracted into IL-mE medium. The phase separation of stable IL-mE was carried out by the micellar cloud point extraction approach. The influence of of different parameters such as pH, oxine concentration, centrifugation time and rate were investigated. At optimized experimental conditions, the limit of detection and enhancement factor were found to be 0.132 mg/L and 70 respectively, with relative standard deviation o5%. In order to validate the developed method, certified reference materials (SLRS-4 Riverine water) and human serum (Sero-M10181) were analyzed. The resulting data indicated a non-significant difference in obtained and certified values of Cu2 þ . The developed procedure was successfully applied for the preconcentration and determination of trace levels of Cu2 þ in environmental and biological samples. & 2015 Elsevier Inc. All rights reserved.

Keywords: Copper Ionic liquid Dispersive liquid–liquid microextraction Phase inversion method Cloud point extraction Flame atomic absorption spectrometry

1. Introductıon Copper (Cu2 þ ) is an important essential element and is associated with number of metalloproteins (Vulpe and Packman, 1995; Milne, 1999; Kazi et al., 2010). The major functions of Cu metalloproteins are oxidation–reduction reactions, as Cu-containing enzymes bind and react directly with molecular oxygen. A number of pathological conditions have been attributed to the loss of cupro-enzyme activity (Harris, 1992). The long term high exposure of Cu causes potentially adverse effects to human health. In the presence of cellular reductants, low-molecular-weight Cu2 þ n

Corresponding author. E-mail addresses: [email protected] (S.A. Arain), [email protected] (T.G. Kazi), [email protected] (H.I. Afridi), [email protected] (M.S. Arain), [email protected] (A.H. Panhwar), [email protected] (N. Khan), [email protected] (J.A. Baig), [email protected] (F. Shah). http://dx.doi.org/10.1016/j.ecoenv.2015.12.035 0147-6513/& 2015 Elsevier Inc. All rights reserved.

compounds may play a catalytic role to initiate the free radical reactions. The resulting oxyradicals have the potential to damage cellular lipids, nucleic acids, proteins, and carbohydrates (Kalkan et al., 2002). During infections or inflammatory stress, serum Cu2 þ concentration increases due to acute-phase activity of interleukin 1 (Meng and Zhang, 2006). The Cu2 þ is also a hepato-toxic element because its accumulation in fibrotic liver caused by the HCV infection may contribute to hepatic injury (Arain et al., 2014a; Hatano et al., 2000). Flame atomic absorption spectroscopy (FAAS) has been widely used for the determination of trace quantities of metal ions in biological and environmental samples because it is a relatively simple technique, with high sample throughput and inexpensive equipment (Arain et al., 2014b). However, the direct determination of metals at trace level by FAAS is limited not only by insufficient sensitivity, but also by matrix interference especially in biological samples (Tabrizi, 2007; Duran et al., 2007). Sample preparation plays an important role in analytical process to concentrate and separate the target analytes as well as decrease the interferences

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from the complex matrix samples. A number of sample pretreatment methods have been established for the determination of trace level of copper from different types of samples. Various preconcentration methods including liquid–liquid extraction (Farajzadeh et al., 2009), coprecipitation (Tuzen and Soylak, 2009), cloud point extraction (Shokrollahi et al., 2008), and solid phase extraction (Bulut et al., 2007) have been proposed for preconcentration of trace elements. Liquid–liquid extraction (LLE) is one of the convenient and simple separation tools used for the metals extraction (Pena-Pereira et al., 2009). These methods, despite of their advantages, suffer from limitations, such as significant chemical additives, solvent losses, complex equipment, large secondary wastes, unsatisfactory enrichment factors and high time consumption, that limit their application (Komjarova and Blust, 2006; Ghaedi et al., 2007; Duran et al., 2008). A new trend in analytical chemistry is miniaturization of preconcentration techniques to reduce the consumption of reagents and decrease waste generation (Asghari et al., 2014) Recently dispersive liquid liquid microextraction (DLLME) has been attracted much attention due to its simplicity, rapidity, low sample volume, high recovery and enrichment factor (Rezaee et al., 2006; Baharand Zakerian, 2012). But, like other analytical methods, DLLME also has some limitations, the use of highly toxic extractive and dispersive solvents, which in addition to their toxicity can decrease the partition coefficients of analytes in extraction solvents (Farajzadeh et al., 2014; Skrlikova et al. 2011). In this sense, substantial interest has been manifested on the usage of ionic liquids (ILs) as the green solvent to replace the conventional organic solvents in order to extract heavy metal ions and other pollutants (Ho et al., 2013). These drawbacks of DLLME can be overcome by using them in the form of microemulsion (mE). Ionic liquid based microemulsiondispersive liquid–liquid microextraction (IL-mE-DLLME), as a new class of extraction media and efficient separation tool, not only overcome the solubility limitations of ionic liquids in a polar solvents but also provide hydrophobic or hydrophilic nano-domains there by expanding their potential in micro-heterogeneous systems use for separation and extraction technique (Gao et al., 2007). ILs are organic salts, non-molecular solvents which exist in the liquid state at room temperature, also termed as room temperature ionic liquids (Shah et al., 2012). A number of extraction methods have been reported in which RTILs have been efficiently employed for the extraction of metal ions (Liu et al., 2005). The micro-emulsion (mE) is defined as a thermodynamically stable, clear and isotropic dispersion of one liquid into another immiscible liquid, stabilized by a third component, which can be a surfactant (detergent molecules) or a co-surfactant (alcohol or amine molecules (Friberg, 2007; Behera et al., 2007)). Two main methods have been used to prepared mE: Phase titration and Phase inversion methods. Phase inversion method (PIM) depends upon addition of an excess of the dispersed phase or in response to temperature. The PIM method simply involves titrating water into a mixture containing ionic liquid and surfactant, which initially leads to the formation of a water-in-ionic liquid (H2O /IL) emulsion, then after stirring, it inverts into an ionic liquid-in-water emulsion (IL/H2O) (Shafiq-un-Nabi et al., 2007; Talegaonkar et al., 2008) The aim of present study was to develop an efficient, rapid and environmental friendly ionic liquid based mE-DLLME method for enrichment of trace levels of Cu2 þ in biological (serum) and environmental (water) samples. In the developed method the mE consisting of hydrophobic ionic liquid in-water (IL/H2O), was formed by the phase inversion method (PIM). In which water is used as dispersive media to disperse ionic liquid, while surfactant (TX-100) was used to stabilize the mE by reducing the hydrophobic IL/H2O interfacial tension. The Cu2 þ preconcentration was

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mediated by chelation with the 8-hydroxyquinoline (oxine) reagent, followed by extraction with IL-mE. Separation of IL-mE from the aqueous phase could be induced by the miceller cloud point extraction technique. The effect of various experimental parameters on the IL-mE-DLLME were investigated and optimized. The validity of proposed method was checked by analyzing trace levels of Cu2 þ in certified reference materials (water and serum), drinking water and blood serum samples of adolescent female HCV patients along with the healthy referents.

2. Experimental 2.1. Chemicals and reagents Ultrapure water obtained from ELGA lab water system (Bucks, UK), was used throughout the work. Certified standard solution of Cu2 þ (1000 mg/L) was obtained from the Fluka Kamica (Bush, Switzerland). Working standard solutions were obtained by appropriate dilution of the stock standard solutions before analysis. Concentrated HNO3 (65%) and H2O2 (30%) were purchased from Merck (Darmstadt, Germany). 1-Butyl-3-methylimidazolium hexafluorophosphate [C4mim][PF6] was purchased from Sigma-Aldrich (Germany). The oxine was obtained from (Merck), prepared by dissolving appropriate amount of reagent in 10 mL ethanol (Merck) and diluting to 100 mL with 0.01 M acetic acid and were kept in refrigerator at 4 °C for one week. The nonionic surfactants Triton X-114 and Triton X-100 were obtained from Sigma (St. Louis, MO, USA). The 0.1 mol/L acetate and phosphate buffer was used to set the pH of the solutions. The pH of the samples and standards were adjusted to the desired pH by the addition of (0.1 mol/L HCI/ NaOH) solution in the buffer. All glassware used in the experiments were cleaned with pure water, soaked in 2.0 mol/L of HNO3 and washed with ultrapure water to avoid contamination. 2.2. Instrumentation A Perkin-Elmer Model AAnalyst 700 (Norwalk, CT) flame atomic absorption spectrophotometer was used. The Cu2 þ hollow cathode lamp was run under the conditions suggested by the manufacturer. A single element hollow cathode lamp was operated at 7.0 mA and spectral bandwidth of 0.7 nm. The analytical wavelength was set at 324.8 nm. The acetylene flow rate and the burner height were adjusted in order to obtain the maximum absorbance signal. A pH meter (Ecoscan Ion 6, Malaysia) was employed for pH adjustments. Centrifugation was carried out by using Model-1465 centrifuge (speed range 0–6000 rpm, timer 0–60 min, 220/50 Hz, HISTAM-R, Spain). Optical microscopy was used to study the microstructure of selected samples (Hund wetzlar D-35580, medprax, germany) with a 60*0.25 objective lens. Turbidity of all the formulated mEM was analyzed by measuring its absorbance at a wavelength of 600 nm (UV–vis Spectrophotometer Biochrom libraS22, Cambridge, UK). 2.3. Sample collection and pretreatment procedure The blood samples were collected from 90 adolescent girls have hepatitis C (HCV), attending the outpatient clinic and admitted to the hepato-gastroenterology ward at the Civil Hospital in Hyderabad, Pakistan. The HCV patients were tested by anti-HCV Antibodies test (positive RNA test /PCR test). For comparative purpose 75 healthy girls of same age group (12–15 years) as referents (mostly the relatives of patients), were also selected. They all were residents of Hyderabad and different areas of Sindh, Pakistan. At the start of the study, weight, height, blood pressure, and

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biochemical data of the participants were measured and recorded. All patients and referents provided a written consent, confirming that they accepted conditions to be donated blood sample and they were informed about the whole experimental procedures. Prior to the biological sample collection, they have undergone a routine medical examination. A questionnaire was administered to all patients and referents to collect details of their physical data, ethnic origin, dietary habits, age, and consent. The criteria for the selection of referent subjects were based on same age group, socioeconomic status, residential areas, and dietary habits to patients. The study protocol was approved by the local ethics committee of Sindh University Jamshoro and Higher Education Commission of Pakistan. The venous blood samples (5 mL) were collected from patients and referents, using metal-free safety heparinized vacutainer s blood collecting tubes (Becton Dickinson, Rutherford s, USA). About 2 mL of blood samples were sent to the hospital pathological laboratories for biochemical tests using standard methods. Remaining (3 mL) samples were used for separating the sera. The blood is allowed to clot at room temperature for 15–30 min, then centrifuged for 5–10 min at 2500 rpm. The supernatant fluid was separated by a Pasteur pipette, labeled, and stored at  20 °C until analysis. The drinking tap water samples were collected on alternate months in 2013 from domestic treated water supply to Hyderabad city, Pakistan. The tap water was allowed to run for 10 min and approximately 1000 mL of water was collected (n ¼100) in a beaker. All water samples were filtered through a 0.45 mm pore size membrane filter (Millipore Corporation, Bedford, MA, USA) immediately after sampling to remove suspended particulate matter. The pH of all water samples was checked with a pH meter. 2.4. Sample preparation Triplicate samples of serum (0.2 mL), of each patient, referents and replicate six samples of certified materials (serum) were taken in separate polytetrafluoroethylene (PTFE) flasks. About 2 mL of a freshly prepared mixture of concentrated HNO3–H2O2 (2:1, v/v) were added to each flask and kept for 10 min at room temperature. The contents of flasks were digested in a microwave untill a semidried mass was obtained. Complete digestion of samples required 2–3 min. The contents of the flask residues were dissolved in 0.1 mol/L HNO3 and made up to volume mark in volumetric flask (25 mL in capacity). All experiments were conducted at room temperature (30 °C). 2.5. Preparation of ionic liquid microemulsion (IL-mE) The PIM have been used to prepare IL-mE by titrating an aqueous phase into a surfactant-ionic liquid mixture with constant stirring at room temperature. The ionic liquid was added to surfactant at different ratios ([C4mim][PF6]:TX-100) in the PTFE flasks and mixed them, using a magnetic stirrer ( 400,800 rpm), then titrated against the drop wise addition of water for 10–30 min until a transparent and homogenous mE ([C4mim][PF6]: TX-100/H2O) was formed. The obtained mE was optimized by taking 100 mL of [C4mim][PF6] and 200 mL of TX-100 at (0.5:1.0) ratio by titrating with 5 mL of water at 700 rpm for 30 min, and stored at 25 °C to check the stability then applied successfully to the proposed method. 2.6. Ionic liquid assisted microemulsion dispersive liquid liquid microextraction (IL-mE-DLLME) procedure The 10 mL aliquots of Cu2 þ standard solution of in the concentration range 2.5–250 μg/L and acid-digested duplicate serum

samples of each patients and control subjects, while 25 mL of drinking water samples were transferred into a conical bottom glass centrifuge tube. Added 500 mL (2.0  10  3–10.0  10  2 mol/L) of oxine and pH was adjusted in the range of 2–10 using 0.1 mol/L of NaOH/HCl. Then gradually added 500–1500 mL of synthesize IL-mE, to entrap the hydrophobic chelate of Cu-oxine from aqueous medium. As the synthesize IL-mE was thermodynamically stable and did not show phase separation after the centrifugation. Separation of IL-mE from aqueous phase could be induced by miceller cloud point extraction technique. So in 2nd extraction phase 50 mL of 1% Triton X -114 was added to entrap Cuoxine complex from extraction media. The contents of tubes were kept on thermostatic bath at 55 °C for 2–5 min. The seperarion of the phases were accelerated by centrifuging for 5 min at 3500 rpm. After cooling in an ice bath for 2–5 min, the ionic liquid-surfactant rich phase became viscous, and was retained at the bottom of the tube. The supernatant aqueous phase was discarded, and the remaining ionic liquid-surfactant phase was diluted with 0.5 mL of acidic ethyl alcohol (0.1 M HNO3), and finally subjected to FAAS for analysis.

3. Result and discussion 3.1. Characterization of IL-mE The morphology of IL-mE was evaluate through visual assessment and using optical light microscopy. The Fig. 1(a) and (b) shows the visual appearance of IL-mE which is changed from milky white to optical transparent and no phase separation has been seen which shows that formulation of mE is stable. The optical microscope image of IL-mE (Fig. 1(b)) indicates the dispersion of IL micro droplets in a continuous water phase. The turbidity of mE was analyzed by measuring the absorbance of mE at a wavelength of 600 nm using UV–visible spectrophotometer. Experiments were conducted to point out the stability of IL-mE at different interval of time from 1 h to 170 h. The stability of IL-mE was checked by simple visual inspection and visible spectrometry at 600 nm at different time interval at 25 °C. It was observed that the absorbance value was increased upto 8% i.e become slightly turbid after one weak (170 h), which represent the change in the morphology of mE droplets from optically transparent to milky white and indicates the stability of mE in the desired condition for sufficient time period. The driving force of IL-mE formation is considered to be the hydrogen bonding between C2_H of bmim þ and ethoxy/hydroxyl _ _ O , between PF6  and H  of the TX-100 hydroxyl terminal, and dipole-induced dipole type of interaction between TX-100 phenyl π-cloud and bmim þ . Reduced turbidity of samples can be attributed to minimum droplet size that results in comparatively weak scattering and making the formulated mE system optically transparent (Bumajdad and Eastoe, 2004). 3.2. Effect of pH The separation of metal ions by proposed method involves prior formation of a complex with sufficient hydrophobicity to be extracted into the small volume of the sedimented phase. The pH plays an important role in metal–chelate formation and its subsequent extraction. A series of experiments were performed by adjusting the pH from 2 to 10 using hydrochloric acid/sodium hydroxide. It was shown in Fig. 2(a) that the extraction was almost quantitative at pH range of 6.5–7.5. Such a neutral pH range provided convenience for the (IL-mE-DLLME) operation on aqueous samples. Extraction recovery of Cu2 þ will decrease at (8 4 pH and

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Fig. 1. Photographs and Optical microscopy images of the IL-mE-DLLME prepared using PIM method at different interval of time (a) 1 h (b) after 170 h.

Fig. 2. (a). Effect of pH on the % recovery of Cu2 þ using IL-mE-DLLME. Conditions: Cu2 þ :10 mg/L, ligand (oxine) 5  10  3 mol/L, IL-mEM:1000 μL, Centrifuging time: 5 min, Centrifuging rate: 3500 rpm. (b) Effect of oxine concentration on the % recovery of Cu2 þ using IL-mE-DLLME. Conditions: pH 7.0, 10 mg/L Cu2 þ , 1000 μL IL-mEM, Centrifuging time: 5 min, Centrifuging rate: 3500 rpm. c. Effect of IL-mEM volume on the % recovery of Cu2 þ using IL-mE-DLLME: pH 7.0, 10 mg/L Cu2 þ , ligand (oxine) 5  10  3 mol/L, Centrifuging time: 5 min, Centrifuging rate: 3500 rpm.

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Table 1 Validation of methodology using certified refrence materials and standard addition method in a real water sample. Certified samples

Experimental value

Certified value

Water (SRM 1643e) mg/L

22.6 7 1.6a (99.3 7 1.2)b

22.8 70.31

Human serum SeroM10181 (mg/L)

1.19 70.09 (99.2 7 0.8)b

1.20 7 0.07

Standard addition/recovery of Cu2 þ in a drinking water sample (sample volume 25 mL, n¼ 6) Added (lg/L) 0 2.5 5 10 a b

Measured (lg/L) 17.4 7 1.6 20.3 70.44 22.4 70.48 26.6 70.52

Recovery (%) – 102 100 97

mean 7 standard deviation (n¼ 6), tCritical ¼ 2.57 The values in parenthesis (% recovery)

o5), because Cu-oxine complex is decomposed in acidic medium and get precipitated at basic medium. So, pH 7.0 was chosen for further experiment. 3.3. Effect of oxine concentration In this study oxine was used as a ligand due to the hydrophobic nature of its metal chelates. The effect of oxine concentration on the recovery of analyte was evaluated in the range of 2.0  10  3–10.0  10  2 mol/L. The results are shown in Fig. 2 (b) indicate that the quantitative extraction efficiency for Cu2 þ ions increases up to 5.0  10  2 mol/L of oxine and above this concentration there is no significant effect on the extraction efficiency of developed procedure. Therefore, 5.0  10  2 mol/L of oxine was selected for the subsequent experimental work. 3.4. Volume of extractant (synthesized IL-mE) To study the effect of extractant (IL-mE) volume, it was used in the range 500–1500 mL (containing 10–30 mL of IL). Fig. 2(c) shows the effect of extractant on the extraction recovery of Cu2 þ . It was observed that optimum level of Cu2 þ was extracted when volume of IL-mE was 4 900 mL. Hence 1000 mL of IL-mE (containing 20 mL of IL) was selected for the subsequent experimental work. 3.5. Salt effect The influence of ionic strength on the efficiency of microextraction of the proposed (IL-mE-DLLME) procedure was investigated by adding different concentrations of NaCl (0.1–0.5 mol/ L). The obtained data indicated that the extraction process was improved due to salting out effect at 0.2 mol/L of salt and decreased at higher concentration. Thus 0.2 mol/L of salt concentration was use used for the following experimental work.

3.7. Effect of the co-existing ions The study was performed using 10 mL standard solutions containing 25 μg/L of Cu2 þ in the presence of various amounts of other ions, and subjected to the preconcentration method as described in Section 2.6. The tolerance limit of ions was fixed as the maximum amount, causing an error not greater than 5%. Among the selected interfering ions, Na þ and K þ at the concentration of 2500 mg/L, Ca2 þ , Mg2 þ , Cl  and SO−4 at the concentration of 2000 mg/L, Cd2 þ 30 mg/L, Pb2 þ 25 mg/L, Mn2 þ , Zn2 þ and Ni2 þ 20 mg/L, while Fe3 þ at the concentration of 10 mg/L did not show any interferences. The obtained results confirmed that the presences of large amounts of coexistent ions commonly present in serum and water samples have no obvious influence on the proposed IL-mE-DLLME method for the determination of Cu2 þ ions. 3.8. Analytical figures of merit The calibration graph using the proposed method was linear with a correlation coefficient (R2) of 0.997 at the concentration range of 2.5–250 mg/L. Regression equation for Cu2 þ with and without IL-mE-DLLME was obtained as, Abs ¼7.6077 (Cu2 þ )  0.0001 and Abs ¼0.1082 (Cu2 þ )þ 0.0049 (R2 ¼ 0.997), respectively. The detection limit (LOD) calculated as under 3 s/m was 0.132 mg/L, where ‘s’ is the standard deviation of ten measurements of the blank and ‘m’ is slope of the calibration graph. The experimental enhancement factor (EF) was calculated as the ratio of slopes of the calibration graphs with and without preconcentration was 70. The analytical characteristics, precision of method, expressed as the % relative standard deviation (% RSD) of a minimum 6 independent analyses containing 10 mg/L of Cu2 þ , was found to be o5%. The validity of proposed method was checked by analysis of Cu2 þ in human serum Sero-M10181 and SRM 1643e (water) certified reference material. The recovery results given in Table 1 show a good agreement between obtained and certified values. The paired t-test was applied to compare the results obtained by IL-mE-DLLME and certified value of Cu2 þ . The texperimental value is lower than the tcritical (2.57) at a confidence interval of 95%, indicated a non-significant difference (p Z0.05). Furthermore, the accuracy of the developed method was evaluated by applying the addition/recovery experiments for a water sample with known standards of Cu2 þ at two concentration levels. The recovery results reported in Table 1 showed a good agreement between the spiked and found values, which indicated the successful applicability of the proposed method for determination of Cu2 þ in real samples. The use of toxic organic solvents (i.e., chloroform, carbon tetrachloride, methanol and acetone etc) (Shrivas and Jaiswal, 2013; Acar and Kara, 2014), as extractants and dispersant has been replaced with green alternatives such as IL, TX-100. Results obtained with our proposed method were compared with the other reported preconcentration methods for Cu2 þ determination (Table 3) (Bumajdad and Eastoe, 2004; Kljajić et al., 2011; Wen et al., 2011; Shrivas, 2010; Mohammadi et al.,2009; Farajzadeh et al., 2008; Anthemidis and Ioannou, 2009). The proposed method has generally low LOD and high enhancement factor is comparable with reported procedures described in the literature, as shown in Table 2.

3.6. Effect of centrifugation time and rate 3.9. Application The effect of centrifugation rate and time upon extraction efficiency was studied in the range of 1500–4500 rpm and 2–10 min, respectively. A centrifugation time of 5 min and 3500 rpm was selected as optimum; since complete phase separation occurred at the end of this period, while at lower or higher centrifuge time and rate, the recoveries were both lower.

The developed method was applied to the preconcentration and determination of trace levels of Cu2 þ in drinking water and serum samples of female adolescent HCV patients. The mean concentrations with standard deviations of Cu2 þ in serum samples are shown in Table 3. The resulted data indicated that high levels

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Table 2 Comparative data of analytical characteristics of present IL-mE-DLLME for Cu2 þ with previous reported preconcentration methods. Method

Techniques

EF

LOD (mg/L)

RSD%

sample

Linear range (mg/L)

Ref.

SDMEa LLEb DLLMEc DLLME IL-DLLMEd SI-DLLMEe DLLME DLLME IL-mE-DLLME

Spectrophotometry Spectrophotometry FAAS FAAS FAAS FAAS UV–vis FAAS FAAS

33 5 – 42–48 – – – 28 70

0.15 2–4 0.5 3.0 0.45 0.04 5 3.4 0.132

3.4 1.8 1.4 5.1 3.3 2.1 1.3–5.4 0.7 3.7

Food and water Water and soil Water Water Water Water … Human hair and tea Human serum and water

5–1000 10–400 1–600 50–2000 2–50 0.16–12.0 20–90 5–200 2.5–250

Bumajdad and Eastoe (2004) Kljajić et al. (2011) Wen et al. (2011) Shrivas (2010) Mohammadi et al. (2009) Farajzadeh et al. (2008) Anthemidis and Ioannou (2009) Present work

a

Single drop micro extraction. Liquid–liquid extraction. Dispersive liquid liquid microextraction. d Ionic liquid-dispersive liquid–liquid microextraction. e Sequential injection ionic liquid dispersive liquid–liquid microextraction. b c

Table 3 Determination of Cu2 þ in serum and tap water samples using proposed IL-mEDLLME method. Drinking water (lg/L) 187 2.65a b Serum (mg/L) Referents (n ¼75) bHCV patients (n¼ 90) p-value 0.943 70.33a 1.53 70.25 o 0.001 a b

mean 7 standard deviation (x7 s). adolescent females (patient and referents).

of Cu2 þ was observed in the serum samples of HCV patients as compared to age matched healthy referent subjects (po 0.01). The elevated level of Cu2 þ in serum samples of female adolescent HCV patients was found at 95% confidence intervals [CI: 1.451–1.505 mg/L] versus referents [CI: 0.785–1.009 mg/L]. The unpaired Student's t-test between the levels of Cu2 þ in serum samples of referents and HCV patients at different degrees of freedom and probabilities were calculated. Our calculated tvalue exceeds that of tcritical (1.96 7 0.2) value at the 95% confidence intervals, which indicated that the values of Cu2 þ in referents and patients, have significant differences (p ¼0.01–0.001). The pH of tap water using for drinking and other purposes were found in the range of 6.9–7.5. The levels of Cu2 þ in drinking water samples from municipal water supply system was found to be in the range of 14.2–25.4 mg/L. Levels of copper in running or fully flushed water tend to be low where as those of standing or partially flushed water samples, it can be substantially higher. Copper works its way into the water by dissolving from copper pipes in the household plumbing. The longer the water has stood idle in the pipes, the more copper is likely to be dissolved (Dietrich et al., 2004). The concentration of Cu2 þ is very low in water samples as compared to WHO recommended value (2000 mg/L). So there is no correlation between Cu2 þ in drinking water and hepatitis. As Cu2 þ accumulation in hepatitis patients is due to its metabolism in human body during viral infection such HCV. It was reported that the hepatic Cu2 þ contents increased with the progression of hepatic fibrosis, and its presence may enhance HCV infection (Hatano et al., 2000). It was also studied that elevated levels of Cu2 þ in sera and have also suggested that this may probably reduce the defense strategies of the organism (Kalkan et al., 2002).

4. Conclusion A novel green miniaturize dispersive ionic liquid based microemulsion method IL-mE-DLLME coupled with FAAS was first time introduced for preconcentration and determination of Cu2 þ

in drinking water and serum samples of female adolescent HCV patients. The extraction recovery of metal ions by mE is obviously advantageous as compared to the traditional treatment processing, related to pollution with toxic organic solvents. The mE extraction system can effectively accelerate the extractability in terms of the spontaneous formation of the microemulsion structure as well as the enormous micro-interfacial surface area. Higher Cu2 þ content in serum samples of adolescent HCV patients may contribute to hepatic injury by oxidative stress.

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