Accepted Manuscript
Cloud point assisted dispersive ionic liquid –liquid microextraction for chromium speciation in human blood samples based on isopropyl 2- [(isopropoxycarbothiolyl)disulfanyl] ethane thioate
Hamid Shirkhanloo, Mehri Ghazaghi, Mohammad Mehdi Eskandari
PII: S2214-1812(16)30018-0 DOI: 10.1016/j.ancr.2016.10.002 Reference: ANCR 53
To appear in: Analytical Chemistry Research Received Date: 13 July 2016
Revised Date: 12 October 2016 Accepted Date: 14 October 2016
Please cite this article as: H. Shirkhanloo, M. Ghazaghi, M.M. Eskandari, Cloud point assisted dispersive ionic liquid –liquid microextraction for chromium speciation in human blood samples based on isopropyl 2-[(isopropoxycarbothiolyl)disulfanyl] ethane thioate, Analytical Chemistry Research (2016), doi:
10.1016/j.ancr.2016.10.002.
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Cloud point assisted dispersive ionic liquid –liquid microextraction for chromium speciation in human blood samples based on isopropyl 2-[(isopropoxycarbothiolyl)disulfanyl] ethane thioate
Hamid Shirkhanloo1, 2,, Mehri Ghazaghi3 and Mohammad Mehdi Eskandari1
1Research Institute of Petroleum Industry (RIPI), West Entrance Blvd., Olympic Village, P.O. Box:
14857-33111, Tehran, Iran
2Occupational and Environmental Health Research Center (OEHRC), Iranian Petroleum Industry Health Research Institute (IPIHRI), PIHO, Tehran 14665-1137, Iran
3Department of Chemistry, Semnan University, Semnan 35131-1911, Iran.
Corresponding author: Hamid Shirkhanloo Email: [email protected]
Address: Department of Chemistry and Environment, RIPI, Tehran, Iran Tel: (+98) (021) (48251)
Fax: (+98) (021) (66701474)
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Abstract
A efficient and fast method based on isopropyl 2-[(isopropoxycarbothiolyl)disulfanyl]
ethane thioate (IICDET) were used for the speciation and determination of trace amount of Cr(III and VI) in human biological samples by cloud point assisted dispersive ionic liquid – liquid microextraction (CP-DILLME). Cr(VI) has carcinogenic effects, so, speciation of chromium in human body such as blood cells is very important. The cloudy solution was achieved by the mixture of acetone and IL ([C8MIM][PF6]) in human blood samples containing Cr(III) ions that were already complexed by IICDET at pH 4.5. After reduction Cr(VI) to Cr(III) by ascorbic acid, chromium speciation was obtained based on total chromium determination by electro thermal atomic absorption spectrometry (ET-AAS) and difference between total Cr and Cr(III) content.. In addition, Cr speciation in human blood cells was calculated based on IICDET/ CP-DILLME and hematocrit blood test (HCT). After optimized conditions, the enrichment factor (EF), Linear range and limit of detection (LOD) was obtained 25.2, 0.02- 1.75 µg L-1 and 5.4 ng L-1 in human biological samples respectively.
The validation of methodology was achieved by certified reference material (CRM) and ICP- MS technique.
Keywords: Chromium, Speciation, Isopropyl [(IsopropoxyCarbothiolyl) Disulfanyl] Ethane Thioate, Cloud point assisted dispersive ionic liquid liquid microextraction, Hematocrit, Human blood.
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1. Introduction
Recently, heavy metals get distinguished from other toxic pollutants which, due to their non-biodegradability can accumulate in living tissues, thus becoming concentrated throughout the food chain and can be readily absorbed by the human body. Even a very small amount of them can cause severe physiological or neurological damage to the human body.
Chromium is a major pollutant for the environment, usually as a result of some industrial pollution including tanning factories, steelworks, industrial electroplating and wood preservation. Among the elements that currently need to be determined in environmental and pharmaceutical products, chromium is important in view of some health risks such as chromosomal aberration, mutations, carcinogenicity, and transformation in cultured cells and a variety of DNA lesions [1-3].
Chromium species exist mainly in two different oxidation states in the environment, Cr(III) and Cr(VI), which have contrasting physiological effects. Cr(III) compounds play an important role in the metabolism of glucose and certain lipids; in addition, it is considered an essential trace element for the maintenance of an effective protein metabolism in humans. [4- 6] On the contrary, the Cr(VI) toxic and carcinogenic effects present are due to their strong oxidation potential and their relatively small size, which enables them to penetrate through biological cell membranes, providing damage to macromoleculessuch as proteins and DNA.
Cr(VI) inhibits the enzymatic sulfur uptake of the cell and is also harmful to lungs, liver and kidneys [7-9].
Chromium can enter the human body through breathing or drinking water, and the level of it in the air, water and biological samples is very low. Chromium concentration in drinking water is generally less than 2 µg L-1 [10]. The World Health Organization (WHO)
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states that the guideline values of 50 µg L Cr(VI) are considered to be too high as compared to its genotoxicity, and the national health and nutrition examination survey (NHANES) claims normal chromium levels found in blood are 0.1 - 1.7 µg L-1 and 0.24 - 1.8 µg L-1 in urine [11-13]. Cr(VI) compounds, once inside the bloodstream, are actively transported into red blood cells (RBC) via nonspecific anionic channels and then rapidly reduced to Cr(III) which becomes bound to hemoglobin in RBCs (Cr6+→Cr5+→Cr4+→Cr3+). Therefore, chromium levels in the red blood cells indicate exposure to Cr(VI) and can be changed to Cr(III) within these cells [14]. The reactive species produced by the reduction of Cr(VI), particularly Cr(III), can form both ionic and coordinate covalent complexes with DNA as inter strand cross-links (DNA-Cr-DNA) . In order to provide a timely warning of chromium exposure, it is highly desirable to develop suitable procedures for chromium speciation.
Sensitive techniques for determination of chromium species include ion chromatography, inductively coupled plasma mass spectrometry [15], luminescence quenching [16], stripping voltammetry[17], co-precipitation [18, 19], flame atomic absorption spectrometry (F-AAS) [20], neutron activation analysis (NAA) [21], inductively coupled plasma optical emission spectrometry (ICP-OES) [22, 23], inductively coupled plasma-mass spectrometry (ICP-MS), [24] ion chromatography inductively coupled plasma-mass spectrometry (IC- ICP-MS) [15] and electrothermal atomic absorption spectrometry (ETAAS) [25, 26] analysis techniques that were frequently coupled with a prior preconcentration and/or separation steps. However, the high instrumental and operational costs and the high detection limits are common disadvantages of many of these methods. Sample preparation procedures such as liquid-liquid extraction (LLE) [27, 28], homogeneous liquid-liquid extraction [29, 30], solid phase extraction (SPE) [4, 31], liquid-phase microextraction (LPME) [32] and cloud point extraction (CPE) [33] are developed to simplify analytical approaches as it reduces costs.
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Dispersive liquid-liquid micro-extraction (DLLME) is a miniaturization of the traditional LLE technique, where the extraction phase is a drop of a few microlitres of a water-immiscible solvent that can be directly immersed in the sample and dispersed by organic solvent.
Although organic solvents (i.e., octanol, cyclohexane, and toluene) are useful as extraction phase, recently the use of ionic liquids (IL) has been proposed in LLE. They have various advantages over traditional organic solvents, such as low vapour pressure, high stability, large viscosity, adjustable miscibility and polarity, good extractability for different organic and inorganic compounds [34-37].
Cloud point extraction (CPE) utilizes the clouding behavior of a solution that containing a nonionic surfactant is heated before being allowed to settle. Because the surfactant is dehydrated during the settling process, the liquid separates into aqueous and surfactant-rich phases. The cloud point phenomenon has been used in separation science for extraction, purification and preconcentration [38, 39].
The aim of this work is speciation of Cr(III) and Cr(VI) in human blood cells, whole blood and serum samples (35 exposed subjects and 35 unexposed controls) based on IICDET by CP-DLLME. Experimental parameters affecting the extraction process were optimized and the performance of the proposed method was evaluated.
2. Materials and methods
2.1. Apparatus
Determination of chromium was performed with a spectra GBC electro-thermal atomic absorption spectrometer (ET-AAS, Plus 932, Australia) using a graphite furnace module (GF3000, GBC). The operating parameters for the metal of interest were set as recommended by the manufacturer. A hollow cathode lamp (GBC) operating at a current of 6 mA and a wavelength of 357.9 nm with a spectral bandwidth of 0.2 nm was used. The
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working range, mode and volume injection have obtained 0.5- 45 µg L , Peak Area and 20 µL, respectively. The pH values of the solutions were measured by a digital pH meter
(Metrohm 744). A micro-hematocrit instrument (Jouan, Italy, A13-HCT) was used for determination of the ratio of blood cells/serum (HCT %) in whole blood samples. Inductivity coupled plasma mass spectrometry (ICP-MS) was used for determination of ultra-trace chromium in standard and human blood samples (Perkin Elmer, QP, Elan6000 DRC, RIPI, Iran).
2.2. Reagents and Materials
All reagents with ultra-trace analytical grade were purchased from Merck Germany.
Iodine, dichloromethane (CH₂Cl₂), dithiocarbomate (CH2NS2), sodium thiosulfate (Na2S2O3),
magnesium sulfate (MgSO4) and hexane (C₆H₁₄) were prepared from Merck. Cr(III) stock solution was prepared from an appropriate amount of the nitrate salt of this analyte as 1000 mg L-1 solution in 0.01 mol L-1 HNO3 (Merck). Cr(VI) stock solution was prepared from an appropriate amount of the 1 g of Potassium salt (K2CrO4) of this analyte as 1000 mg L-1 solution in 1 % HCl. Standard solutions were prepared daily by dilution of the stock solution. The pH adjustments were made using appropriate buffer solutions including sodium phosphate (H3PO4/NaH2PO4, 0.1 molL-1) for pH 2-3, ammonium acetate (CH3COOH/
CHCOONH4, 0.1 mol L-1) for pH 4-6, sodium borate (NaBO2/HCl, 0.1 mol L-1) for pH 7,and ammonium chloride (NH3/NH4Cl, 0.1 mol L-1) for pH 8-10 (Merck). Polyoxyethylene octyl phenyl ether (TX-100, CAS N: 9036-19-5) as the anti-sticking agent was purchased from Merck. Ultra-pure lithium heparin (H0878, CAS N: 9045-22-1, 100KU) and sodium citrate (S4641, CAS N: 6132-04-3, 25 g) were purchased from Sigma-Aldrich in Iran. Isopropyl 2- [(isopropoxycarbothioyl) disulfanyl] ethane thioate was synthesized and purified in RIPI laboratories (IICDET; (CH3)4 (CO)2 S4). Ultrapure water (18 MΩ.cm) was obtained from
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Millipore continental water system (Bedford, USA), and 1-octyl-3-methylimidazolium hexafluorophosphate was prepared from Sigma Aldrich (Germany, C12H23N2·PF6, CAS N:
304680-36-2, 5g). Graduated micro centrifuge conical tubes with cap (2-10 mL) were purchased from Sigma-Aldrich (Germany, Product N: SIAL311GZ2F).
2.3. Sampling
For sampling, all glass tubes were washed with a 0.5 mol L-1 HNO3 solution for at least 24 h and thoroughly rinsed 6 times with ultrapure water before use. As chromium concentrations in whole blood and serum are very low, even minor contamination at any stage of sampling, sample storage and handling, or analysis has the potential to affect the accuracy of the results. Heparin is commonly used as anticoagulants in human blood samples.
The blood collection tube with heparin was aliquoted into Eppendorf tubes (5 mL) and kept at -20OC for one week. For analysis in whole blood, 10 µL of pure heparin (free chromium) is added to a 10 mL blood sample of painting workers from Iranian car factory, Tehran, Iran.
Hematocrit percentage (HCT %) was determined by centrifuging heparinized blood in a capillary tube (micro-hematocrit tube) at 10000 rpm for 8 minutes. The human blood sample was maintained at –20 °C in a cleaned glass tube. Serum and blood samples were collected from exposed (35) and unexposed (35) subjects from Iranian car factory between 20 to 45 years of age.
2.4. Synthesis of isopropyl 2-[(isopropoxy carbothioyl) disulfanyl] ethanethioate (IICDET)
A solution of potassium O-isopropyl (ditiocarbamate) (1 mmol) in 20 ml of water was cooled in an ice bath; then a solution of iodine (1 mmol) and potassium iodide (1 mmol) in water (20 mL) was added dropwise. After stirring for 1h, the aqueous phase was extracted
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with CH2Cl2 (3 × 10 mL), washed with 30 mL of 10% aqueous Na2S2O3 and then with 30 ml of de-ionized water. The organic layer was dried with anhydrous calcium chloride and evaporated under vacuum. Further purification was achieved by recrystallization in hexane. A pale yellowish crystal of IICDET with a yield of 90% was obtained. The structure and purity of IICDET were confirmed by NMR and IR spectroscopic methods (Fig. 1-3). 1H NMR (CDCl3). δ (ppm): 1.43 (d, 12H, CH3), 5.63 (m, 2H, CH). 13C NMR (CDCl3). δ (ppm): 22.2, 80.6, 207.1. IR (KBr). νmax (cm−1): 2979.8 (s), 2869.9 (w), 1463.9 (s), 1442.7 (s), 1373.0 (s), 1271.1 (s, b), 1145.6 (s), 1082.2 (s), 1048.0 (s, b) 898.8 (s), 796. 5 (s), 690. 5 (m).
Fig. 1 Fig. 2 Fig. 3 2.4. General procedure
A pre-concentration procedure based on IICDET by CP-DILLME was performed as follows: first, 0.15×10-6 mol L-1 of IICDET solution as a ligand, 0.01 mL of triton X-100 (0.3
% w/v) and a 1 mL buffer solution (pH =4.5) were added to 5 mL of human blood samples after dilution with DW up to 5 mL (1:5). Then, cloud point extraction for Cr(III) ions was achieved by adding, 0.12 g (100 µL) of IL ([C8MIM][PF6]) and 0.2 mL of acetone as a dispersive solvent to blood samples. Triton X-100, an emulsifier and anti-sticking agent, was added to the solution in order to raise the efficiency of the extraction procedure. For optimizing and recovery, 5 mL of 0.2 and 1.2 µg L-1 Cr(III) working standard solution was used instead of the blood and serum samples, and 0.12 g of 1-octyl-3-methylimidazolium hexafluorophosphate ([C8MIM][PF6]) dispersed in acetone, was added to standard sample with mixture of IICDET, TX-100 and buffer solution . The cloudy solution was shaken for 2 min by ultrasonic shaking at 25 OC. In order to separate the phases, the turbid solution was
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centrifuged for 4 min at 3500 rpm and the aqueous phase was removed with a transfer pipette.
Cr(III) was back-extracted from IL by adding 0.2 mL of 1.2 mol L-1 nitric acid and the aqueous phase was shaken for 1 min and determined by ET-AAS. In the optimum pH conditions, total chromium was calculated by reduction Cr(VI) to Cr(III) by ascorbic acid solution (0.5 mL, 0.1 mol L-1). Then, Cr(VI) concentration was simply calculated by the difference between total chromium and Cr(III) concentrations. By redox reaction of ascorbic acid, Cr(VI) is reduced to Cr(III) and ascorbic acid is dehydrogenated to dehydroascorbic acid. The reaction can be described by the following equation 1:
Cr2O72−+3C6H8O6+8H+→2Cr3++3C6H6O6+7H2O Eq:1
The blank solutions proceeded the same way and are used for the preparation of the calibration solutions and for measurement of the blanks. Chromium Speciation in blood cells was performed by measuring HCT of blood sample and speciation of chromium in serum and whole blood samples based on proposed procedure. After speciation of chromium in blood and serum samples, level of Cr(III,VI) concentrations in blood cells, were simply obtained by CP-DILLME method. The extraction conditions in IICDET / CP-DILLME method is listed in Table 1.
Table 1
3. Results and Discussion
The proposed method based on IICDET with CP-DILLME was used for chromium speciation in standard solution and human biological matrix (Whole blood, serum and Blood cells) with appropriate accuracy and precision. The results showed us, the mean concentrations of Cr(III and VI) in blood samples of exposed subjects (35N) were significantly higher than unexposed controls respectively [(0.59 ± 0.13 µg/L, 0.27 ±
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0.11µg/L) and (1.29 ± 0.81µg/L, 0.08 ± 0.05µg/L)] and the most Cr (VI) and Cr(III) concentration was existed in blood cells and serum respectively.
The recovery was defined as the percentage of the total analyte (CT) which was extracted into the ionic liquid phase (CIL) (EQ1).
%R= (CIL/CT)×100 (EQ1)
3.1. Effect of ETAAS conditions
In order to increase the accuracy, precision and repeatability, we have used Triton (TX-100 ) in 25 ºC. The one step of temperature program is a drying temperature (125oC) with time of 20 s for water evaporation. Then, a long ramp time of 50 s was chosen as it allowed gradual elimination of trace organic ionic liquid from analyte (600-750 ºC) and avoided chromium loss in this temperature. The influence of pyrolysis temperature on the absorbance was studied up to 1500 ºC. The maximum absorbance was achieved within a range of 1000–
1200 ºC. Therefore, 1100 ºC was selected as the working pyrolysis temperature. The effect of atomization temperature on chromium signal was studied within the range of 2200–2800 ºC, and the maximum signal was obtained at approx. 2500 ºC. Finally, the cleaning time and temperature of graphite tube were ordered at 3 s and 2700 ºC, respectively. The argon flow rate was 300 mL min-1 (Fig. 4).
Fig. 4
3.2. Effect of pH
The influence of sample pH on adsorption of Cr(III) ion on IICDET was investigated using different pH from 1 to 12 for 0.1 µg L-1 Cr(III) as a lower limit of quantification (LLOQ) and 1.5 µg L-1 Cr(III) as upper limit of quantification (ULOQ). The complexation
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was strongly conditioned by the pH of solutions and subsequently affects extraction efficiency of the complex. The results show that the highest extraction efficiency for Cr(III) was achieved in pH 3-5.5, but the recovery values for Cr(VI) were below 5% in the this pH. Thus, the procedure was applied to speciation of two forms of chromium at pH 4.5 (Fig. 5).
The extraction mechanism of Cr(III) ions with IICDET was highly depended to pH solution and complex formation between Cr(III) ions and sulfur groups of IICDET. The sulfur groups can be deprotonated (SH-) at wide range of pH. The extraction capacity of Cr(III) at different pH values may be attributed to the affinities of sulfur groups of ligand for the cationic form of Cr 3+ existing at optimized pH. In addition, the different anionic species of Cr(VI) at low and high pH (pH=2 and pH > 8), namely HCrO4-
, CrO42-
and Cr2O72-
has negatively charged and cannot be adsorbed by negative charge of SH─ group in optimized pH.
Fig. 5
3.3. Effect of ligand concentration
The concentration of isopropyl 2-[(isopropoxycarbothiolyl)disulfanyl]ethane thioate (IICDET) is one of the important parameters which should be optimized by CP-DLLME method. For optimizing, 0.1×10-7─ 0.5×10-6 mol L-1 of IICDET ligand was used in human blood sample by proposed procedure. The results showed that, by increasing ligand concentration up to 0.5×10-6 mol L-1, the recoveries are also increased. Fig. 6 shows that 0.12×10-6 mol L-1 of IICDET was the minimum ligand concentration necessary to achieve maximum extraction efficiency. The signal remained constant from 0.015×10-6 mol L-1 up to at least 0.1×10-6 mol L-1 IICDET for 0.2 µg L-1 of Cr(III) as a LLOQ range. Therefore, 0.2×10-
7 mol L-1 of IICDET concentration was selected. For 1.75 µg L-1 ofCr(III) as a ULOQ range, the signal remained constant from 0.12×10-6 mol L-1 up to at least 0.5×10-6 mol L-1 and 0.15×10-6 mol L-1 was selected as a optimized ligand concentration. The minimum buffer
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concentration required to adjust the pH 3-5.5 and to achieve the best performance of the extraction system was 0.15-0.25 mol L-1.
Fig. 6
3.3. Effect of sample volume and amount of ionic liquid
Sample volume is one of the most important parameters to be studied. The effect of sample volume was examined in different volumes from 1 to 20 mL for 0.2 µg L-1 and 1.5 µg L-1 of Cr(III). Quantitative extraction was observed between 1 to 10 mL. At higher volumes the recoveries are decreased. It was also noticed that higher sample volumes partially solubilized the ionic liquid phase, leading to non-reproducible results. Therefore, a sample volume of 5 mL was selected for further experiments with IICDET - CP-DLLME method (Fig. 7).
It was observed that extraction efficiency of the system was remarkably affected by ionic liquid amount, so it was examined within the range of 0.03–0.2 g. Quantitative extraction was observed at higher than 0.1 g. Therefore, in order to achieve a suitable preconcentration, 0.12 g of ionic liquid ([C8MIM][PF6]) was chosen as optimum leading to a final IL (Fig. 8).
Fig. 7 Fig. 8
3.4. Effect of various mineral acids on back-extraction of ionic liquid phase
Direct injection of ionic liquids into ETAAS was not possible, because ILs have high viscosity. The proposed method was achieved based on back-extraction of chromium from IL with a mineral acidic/basic solution. Therefore, decreasing/increasing of pH, leads to dissociation and releasing of chromium ions into the aqueous phase (mineral acidic/basic solution). Different concentration of mineral reagents from 0.2 ─2 mol L-1 (HCl, HNO3, H2SO4, KOH) were studied for chromium back-extraction from extracting phase (Fig. 9). The
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results showed that 0.5 mol L-1 of HNO3 at 70OC can be back extracted Cr(III) from the IL phase (Fig. 10). After extraction of Cr(III) ions by procedure, 0.2 mL of 0.5 mol L-1 of HNO3 was added in ultrasound batch (1 min, 70 OC) and Cr(III) ions back-extracted from ionic liquid phase to liquid solution, then centrifuging for 2 min and the result solution adjusted to 0.2 mL with DW in micro centrifuge conical tube (2 mL, graduated tube) before determining by ET-AAS with auto sampler.
Fig. 9 Fig. 10 3.5. Effect of ultrasound-assisted extraction time
The optimization of ultrasonication time is crucial to achieve an efficient IICDET / CP-DLLME procedure. In this study, different ultrasound-assisted extraction times ranging from 30 to 240 seconds was evaluated for proposed procedures, respectively. As shown in Fig. 11, by increasing the ultra-sonication time the relative response increases, reaching the maximum value at 110 seconds and then remained constant. Therefore, the ultrasonic times of 2 minutes for ligand complexation (IICDET) before cloudy solution and 1 min for back- extraction of Cr(III) from IL with HNO3 were employed.
Fig. 11 3.6. Effect of matrix
ETAAS is a very specific technique with low sensitivity to interference. Then, the potential interference effects occurring in this procedure are mainly related to the extraction during the pre-concentration step applied to the target samples. Considering the samples of interest, the most probable metal ions’ reported effect of potential interfering ions on the determination of chromium were investigated. The procedure of CP-DLLME was performed using a 5 mL sample containing 1.5 µg L-1 of analyte and 0.2–12 mg L-1 different
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concentration of matrix ions. The tolerate amounts of each ion were the concentration values tested that caused less than 5% of the absorbance alteration. The ions normally present in the sample do not interfere under the experimental conditions used. The results are shown in Table 2.
Table 2 3.7. Analytical figures of merit
Analytical parameters, such as linear range, correlation coefficient, limits of detection, accuracy and precision were evaluated by preconcentrating 5 mL of human biological samples and standard solutions (pH=4.5) containing listed ions. The calibration curve of human biological samples and standard solutions was rectilinear in the range of 0.02 – 1.75 µg L-1 and 0.02 – 1.80 µg L-1, respectively as a lower limit of quantification (LLOQ) and upper limit of quantification (ULOQ). Detection limits and precision (calculated as relative standard deviation) was evaluated for target analyte. The detection limits were calculated as the concentration providing an analytical signal three times higher than the background noaise. The limit of detection (LOD) was obtained 5.4 ng L-1 and 4.6 ng L-1 for 5 mL of human and standard samples, respectively (intra-day, Mean of LOD=5 ng L-1). The precision of the method, expressed as relative standard deviation (RSD %), was calculated from seven individual standards. The RSD(%) of chromium (III) standard solution in different concentrations of 0.02, 0.5 and 1.5 µg L-1 were obtained 4.7, 3.5 and 2.9, respectively (intra- day, mean of RSD% =3.8). The preconcentration factor, calculated as the ratio of the concentration of analyte after preconcentration to that prior preconcentration, was obtained 24.5 and 25.2 for standard and human samples, respectively (intra-day analysis, Mean of PF=25).
3.8. Method validation
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The IICDET /CP-DLLME method was applied to determine Cr(VI)and Cr(III) found as a base value in 5 mL of 35 exposed and control subjects in human serum blood samples (Table 3). The mean concentration of Cr(III) was higher than Cr(VI) in serum subjects, but the mean concentration of Cr(III) in blood samples was lower concentration. The coloration analysis was achieved between Cr(III) and Cr(VI) in subject and control group and there was a correlation (r > 0.3) and regression between Cr(VI) and Cr(III) in blood subjects. The weak correlation (r ≈0.1) in serum and blood cells of exposed subjects were observed. In addition, in control group, no correlation and regression were shown between Cr(VI) and Cr(III) in human samples. The spiked serum and whole blood were prepared to demonstrate the reliability of the method for determination of Cr(III) and Cr(VI). The remaining aliquots were spiked with increasing quantities of Cr(VI)and Cr(III) and analyzed by the IICDET /CP- DLLME method (Table 4). The recovery of spiked samples is satisfactorily reasonable and was confirmed using addition method, which indicates the capability of the system in the determination of Cr(VI) and Cr(III) in human blood samples. The IICDET /CP-DLLME method can be used for speciation and determination of Cr(VI)and Cr(III) in cells of blood samples. Chromium levels in the RBC indicate exposure to Cr(VI) and can be changed to Cr(III) within these cells [14]. Thus, after determining and speciation of concentration of chromium in whole blood and serum samples by procedure, the concentration of Cr(III and VI) in blood cells was simply calculated (Tables 5). For validation, total chromium in real whole blood and blood cells samples prepared with microwave digestion and determined by ICP-MS before/after spiked with standard Cr(VI) and Cr(III) solution. Also, the results of ICP-MS compared to IICDET /CP-DLLME method. The results showed us, the high efficiency and reliability of proposed method for determination and speciation of Cr(VI) and Cr(III) in whole blood, blood cells samples (Table 6).
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Table 3 Table 4 Table 5 Table 6
4. Conclusions
In this research a new method based on IICDET /CP-DLLME was used for the highly speciation and determination of trace amount of Cr(III) and Cr(VI) in blood cells, whole blood and serum samples. Factors influencing in IICDET/CP-DLLME were optimized. It provides a sensitive, efficient, inexpensive and simple method for speciation, preconcentration and separation of the chromium using low sample volumes, which was compatible for handling of biological fluids. The performance of this procedure for quantification of trace levels of chromium in blood samples was satisfactory. The analytical performances of detection of Cr achieved are comparable to reported procedures (Table 7). Finally, the speciation chromium in human biological samples based on IICDET /CP-DLLME with HCT was revealed that the most of Cr(VI) and Cr(III) can exist in red blood cells (RBC) and serum respectively. The IICDET /CP-DLLME method was simply provide the concentration of Cr(III and VI) in human blood cells and it is very important as a toxic and cancer genic agent in human body (DNA-Cr-DNA).
Table 7 Acknowledgements
The authors are thankful to theResearch Institute of Petroleum Industry (RIPI) and Iranian Petroleum Industry Health Research Institute (IPIHRI), PIHO, Tehran, Iran.
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Table 1 Chromium extraction conditions in IICDET /CP-DLLME method
* 5 mL of standard solution and 1 mL of blood samples diluted up to 5 mL with DW (1:5) Value
Parameters
4.5
0.15×10-6 1.0
0.5 0.12 0.2 2.0 4.0 5 pH
IICDET Conc.( mol L-1) Volume of Buffer (mL)
Concentration of HNO3 (mol L-1 ) IL(g)
Volume of Acetone (mL) Shaking time (min) Centrifugation time (min)
*Sample volume (mL)
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Table 2 The effect of matrix ions on the determination of Cr(III) by IICDET method (µg L-1)
This work was performed using 5 mL of 1.5 µg L-1 Cr standard solution.
Ions Maximum tolerance ratio
(matrix ion conc./Cr conc.) Na+, K+, Ba2+, Mg2+, Ca2+, As3+, Co2+, Al3+, Mn2+ 6500
Hg2+, Au3+ 200, 300
As5+, PO43-
, Cl -, CO32-
, SO42-
, NO3-
, F- 7200
Fe3+ and Fe2+
Ag+, Pb2+
Zn2+, Cu2+
Ni2+
2800 250,500 1200
400
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Table 3 Speciation of chromium in serum, blood and blood cells by IICDET /CP─DLLME method (samples analyzed in inter day by blood and serum samples at -20OC) Mean of three determinations of samples ± confidence interval (P = 0.95, n =10)
*Correlations are based on Pearson coefficients (r). Statistical significance will be observed if P < 0.05 Sample
Subjects (n=35) Controls (n=35) Subject
Cr III Cr VI Cr III Cr VI r P value
Serum (µg L-1) 1.06 ± 0.77 0.08 ± 0.04 0.56 ± 0.22 0.04 ± 0.02 0.116 =0.02 Blood Cells (µg L-1) 0.07 ± 0.03 1.25 ± 0.69 0.05 ± 0.02 0.06 ± 0.04 0.072 <0.001 Blood (µg L-1) 0.59 ± 0.13 1.29 ± 0.81 0.27 ± 0.11 0.08 ± 0.05 0.388 <0.005
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Table 4 Validation of chromium speciation based on IICDET /CP─DLLME with standard addition in human serum and blood samples by ET-AAS
Sample
Added(µg L-1) Found (µg L-1) Total Recovery (%)
Cr (III)
Cr (VI) Cr (III) Cr (VI) Cr (III) Cr (V)
Whole Blood
--- --- 0.662 ± 0.026 0.094 ± 0.003 0.756 ± 0.028 --- --- 0.3 --- 0.964 ± 0.048 0.101 ± 0.004 1.065 ± 0.056 101 --- --- 0.2 0.664 ± 0.028 0.289 ± 0.013 0.953 ± 0.045 --- 98
Whole Blood
--- --- 1.002 ± 0.055 0.223 ± 0.021 1.225 ± 0.064 --- --- 0.5 --- 1.477 ± 0.073 0.214 ± 0.011 1.691 ± 0.078 95 --- --- 0.2 0.981 ± 0.041 0.420 ± 0.018 1.401 ± 0.065 --- 98
*Whole Blood
--- --- 0.172 ± 0.009 1.102 ± 0.057 1.274 ± 0.069 --- --- 0.5 --- 0.645 ± 0.031 1.098 ± 0.047 1.743 ± 0.084 95 --- --- 0.5 0.165 ± 0.011 1.584 ± 0.075 1.749 ± 0.092 --- 96
Serum
--- --- 0.655 ± 0.033 0.152 ± 0.006 0.807 ± 0.043 --- --- 0.5 --- 1.137 ± 0.062 0.161 ± 0.005 1.298 ± 0.071 96 --- ---- 0.2 0.653 ± 0.036 0.348 ± 0.016 1.001 ± 0.048 --- 98
Serum
--- --- 1.141 ± 0.058 0.086 ± 0.002 1.227 ± 0.064 --- --- 0.5 --- 1.624 ± 0.077 0.088 ± 0.003 1.712 ± 0.082 97 --- --- 0.2 1.138 ± 0.061 0.289 ± 0.012 1.427 ± 0.078 -- 102 Mean of three determinations ± confidence interval (P = 0.95, n =5)
*Whole Blood (WB) diluted with DW (1:5), the concentration is out of linear range, Final Cr conc. = found Cr conc. × 5 (dilution factor)
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Table 5 Speciation and determination of Cr (VI) and Cr(III) in blood cells based on HCT/ IICDET /CP─DLLME method by ET-AAS (µg L-1)
(1-HCT)×SB (Total Cr) ─ WB (Total Cr)= BC (Total Cr) Cr(VI) ×BC= Cr(VI) ×WB-[(1-HCT) × Cr(VI) SB]
Samples Serum (SB)CP─DLLME Total SB Whole blood (WB)CP─DLLME Total WB Blood Cells (BC) HCT/CP─DLLME Total BC HCT(% ± 0.2)
Cr (III) Cr (VI) Cr (III) Cr (VI) Cr (III) Cr (VI)
Sample 1 1.253±0.052 0.133±0.005 1.386±0.056 0.742±0.025 0.411±0.019 1.153±0.048 0.053±0.002 0.340±0.015 0.391±0.017 45 Sample 2 1.121±0.042 0.213±0.014 1.334±0.058 0.812±0.029 0.644±0.027 1.456±0.061 0.207±0.013 0.529±0.026 0.736±0.028 46 Sample 3 0.953±0.038 0.062±0.002 1.015±0.061 0.541±0.022 0.073±0.003 0.614±0.030 0.045±0.002 0.041±0.001 0.086±0.004 48 Sample 4 0.781±0.032 0.123±0.006 0.904±0.043 0.572±0.028 0.183±0.009 0.755±0.039 0.135±0.006 0.114±0.003 0.249±0.010 44
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Table 6 Comparing of ICPMS method and IICDET /CP─DLLME and HCT/ IICDET /CP─DLLME method for Cr determination in real samples (N=7)
a Based on standard method, blood cell sample after centrifuging ( blood separation from serum) by filtering, acid digestion by microwave and after spike with chromium standard solution determined by ICP-MS.
Sample
ICP-MS (µg L-1) IICDET /CP─DLLME (µg L-1)
Cr III Cr VI Total Cr III Found Cr VI Found Total
Blood
--- ---- 0.752 ± 0.035 0.514 ± 0.026 0.221± 0.011 0.735 ± 0.032 0.50 ---- 1.233 ± 0.037 0.996± 0.049 0.215± 0.010 1.211± 0.055 --- 0.50 1.241 ± 0.041 0.493 ± 0.023 0.705± 0.037 1.198± 0.059
ICP-MS (µg L-1) HCT/IICDET /CP─DLLME (µg L-1)
Cr III Cr VI Total Cr III Found Cr VI Found Total
Cells of blood a
--- ---- 0.517 ± 0.031 0.072 ± 0.005 0.432± 0.025 0.504 ± 0.029
0.50 ---- 0.997 ± 0.044 0.601± 0.036 0.426± 0.027 1.027± 0.058
--- 0.50 1.022 ± 0.046 0.068 ± 0.003
0.939± 0.022 1.007± 0.061
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Table 7 Comparison presented method with published reported method for speciation of Cr in biological samples
a Preconcentration Factor
b Enrichment factor
1 Cloud point extraction
2 Single drop microextraction
3 Diffuse reflectance Fourier transform infrared spectroscopic
4 Task specific ionic liquid-based in situ dispersive liquid–liquid microextraction
5 Directly suspended single droplet microextraction
6 Dual electromembrane extraction
7 Solidified floating organic drop microextraction Method Detection Measurability
of Cr in RBC matrix LOD RSD linear range PF/EF Sample
volume Ref.
CPE1 ETAAS No Human serum 0.02 µg/L 2.6 % - 83.5b 10 mL [40]
SDME2 DRS-
FTIR3 No blood 800 µg/L 2.6 % 5–500 µg/L - 8.0 mL [41]
TSIL-DLLME4 FAAS No water and urine
samples 5.7 µg/L 1.1% 25–750 µg/L 20a 10 mL [42]
DSDME5 ETAAS No Water sample 0.03 µg/L 4.7 % 0.10–2.0 µg/L - 30 mL [43]
DEME6 HPLC No Water sample 5.4 µg/L 9.8 % 20- 500 µg/L 21.8 b 2.1 mL [44]
SFODME7 ETAAS No
water and urine
samples 0.006 µg/L 5.1 % 0.03–0.13 µg/L 327 b 10 mL [45]
SPE ETAAS No Water sample 0.056 µg/L 2.5 % 2.0–160.0 µg/L 25 a 25 mL [46]
IICDET
/CP─DLLME ETAAS Yes
Serum, whole blood, blood
cells
0.005 µg/L 3.8 % 0.02– 1.75 µg/L 25.2a 5.0 mL This work
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Figure captions:
Fig.1. isopropyl 2-[(isopropoxy carbothioyl) disulfanyl] ethanethioate (IICDET)
Fig. 2. 1H-NMR of isopropyl 2-[(isopropoxy carbothioyl) disulfanyl] ethanethioate (IICDET) Fig. 3. 13C-NMR of isopropyl 2-[(isopropoxy carbothioyl) disulfanyl] ethanethioate (IICDET) Fig. 4. Pyrolysis and atomization temperature curves obtained by 20 µL injection of a 1 µg L-1 Cr III solution in presence of HNO3 2 %. Atomization temperature for pyrolysis optimization: 2500 ºC.
Pyrolysis temperature for atomization optimization: 1100 ºC. (95% confidence interval; n =5).
Fig. 5. Optimization of conditions for Cr(III) extraction. The influence of pH on Cr III ─ IICDET extraction in blood and water.
Fig. 6. The influence of IICDET concentration on Cr(III) recovery. Other conditions was as indicated in Table 1 (95% confidence interval; n =5).
Fig. 7. The influence of sample volume on Cr recovery by application of the proposed procedure based on IICDET /CP-DLLME. Other conditions were as indicated in Table 1 (95% confidence interval; n =5).
Fig. 8. The influence of amount of ionic liquid on Cr recovery by application of the proposed procedure. Other conditions of IICDET /CP─DLLME were as indicated in Table 1 (95% confidence interval; n =5).
Fig. 9. The influence of different reagents on back extraction of chromium by proposed method.
Other conditions were as indicated in Table 1 (95% confidence interval; n =7).
Fig. 10. The influence of nitric acid concentration in different temperature for back extraction of chromium from IL by proposed method. Other conditions were as indicated in Table 1 (95%
confidence interval; n =7).
Fig. 11 The influence of ultrasound time with ligand mass on high recovery of Cr(III) by application of the proposed procedure (Mean recovery≈98%). Other conditions of IICDET /CP─DLLME were as indicated in Table 1 (95% confidence interval; n =7).
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Highlights
• Isopropyl 2-[(isopropoxycarbothiolyl)disulfanyl] ethane thioate (IICDET) was synthesized and applied for selective Cr (III) complexation at optimized pH.
•
Cloud point assisted dispersive ionic liquid -liquid microextraction (CP-DILLME) basedon IICDET were used to speciation Cr (III and VI) in human blood
.
• IICDET /CP-DILLME based on hematocrit blood test (HCT) were applied for speciation Cr (III and VI) in human blood cells.
