Research Article pubs.acs.org/journal/ascecg
Development of a Lower Toxic Approach Based on Green Synthesis of Water-Compatible Molecularly Imprinted Nanoparticles for the Extraction of Hydrochlorothiazide from Human Urine Maryam Arabi,† Mehrorang Ghaedi,*,† and Abbas Ostovan‡ †
Chemistry Department, Yasouj University, Yasouj, 75918-74831, Iran Department of Chemistry, Kerman Branch, Islamic Azad University, Kerman, 7635131167, Iran
‡
S Supporting Information *
ABSTRACT: In the present paper, one-pot green syntheses of novel hydrophilic and superparamagnetic molecularly imprinted polymers (MMIPs) for the cleanup and extraction of hydrochlorothiazide (HCT) in human urine are described. The MMIPs were prepared via a sol−gel process using Fe3O4 magnetite as a magnetic component, HCT as a template, tetraethyl orthosilicate (TEOS) as the cross-linker, and 3aminopropyl trimethoxysilane (APTMS) as the functionalized monomer, which could simplify the imprinting process. During the synthesis process, a surfactant was especially used to graft the silica-imprinted nanoparticles. The key step of this research is mild working temperature without consuming any organic solvent during the synthesis of MMIPs in addition to its ability for efficient and highly selective enrichment of HCT in complicated human urine. The morphology, structure, and magnetic properties of the MMIPs were characterized using scanning electron microscopy (SEM), transmission electron microscopy (TEM), Fourier transform infrared spectroscopy (FT-IR), and vibrating sample magnetometry (VSM). The prepared MMIPs were found to be superparamagnetic, which makes them easy to be quickly separated from the sample solution and thus decreases the extraction time. After the dispersive solid phase extraction (d-SPE) by the prepared MMIPs, high performance liquid chromatography (HPLC) was used to determine the target analyte, HCT. Several factors affecting the extraction efficiency, including the pH of the sample solution, the amount of adsorbent, sonication time, eluent, and washing solvent volumes, were evaluated, and the optimum conditions were obtained using the experimental design methodology. Under the optimized conditions, the developed MMIPs-d-SPE linearly responded over 2.5−1000 μg L−1 while a detection limit of 0.75 μg L−1 was obtained. The high selectivity of this method makes it suitable for successful monitoring of analyte in a real sample such as urine with satisfactory recoveries of 90.7−110.0% with the precision of 0.8−6.6%. KEYWORDS: Molecularly imprinted polymers, One-pot synthesis, Water-compatible, Human urine, Magnetic, Hydrochlorothiazide, Experimental design
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INTRODUCTION Hydrochlorothiazide (HCT), 6-chloro-3,4-dihydro-2H-1,2,4benzothiadiazine-7-sulfonamide-1,1-dioxide, is one of the oldest thiazide diuretics of benzothiadiazines widely used in antihypertensive agents, treatment of edema, and management of diabetes insipidus.1−4 The hypotensive effect is believed to be due to the reduction of blood volume by decreasing active sodium and direct relaxation of arteriolar smooth muscle.5,6 It is usually prescribed in dosages of 25−250 mg per day, but incidence of various side effects may occur such as hyperglycemia, hyperuricemia, hyponatremia, and hypokalemia.7 Therefore, various analytical methods, such as square wave voltammetry,8 high performance liquid chromatography (HPLC),9 capillary electrophoresis,10 and spectrophotometry,11 have been reported for quantitative determination of HCT. Most of these methods, in spite of their remarkable advantages, © XXXX American Chemical Society
suffer from drawbacks, including low sensitivity and selectivity, high detection limit, more pretreatment necessary, and requirement to use electroactive nature material,s which encourage us to design and develop new sorbents able to simultaneously extract and preconcentrate the target compound from real samples with complicated matrixes such as urine. Advances in solid phase extraction (SPE) usually refer to improvements in the specific and selective recognition of a particular component, which has a great potential for biological processes. Molecularly imprinted polymers (MIPs) have garnered high attention for their considerable mechanical and chemical robustness, cost-effectiveness, and high selectivity for Received: October 29, 2016 Revised: March 24, 2017
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DOI: 10.1021/acssuschemeng.6b02615 ACS Sustainable Chem. Eng. XXXX, XXX, XXX−XXX
Research Article
ACS Sustainable Chemistry & Engineering a target molecule.12−15 So, it is a superior choice to employ molecularly imprinted materials as adsorbents for extraction. During synthesis, MIPs recognition sites are imprinted in the polymer matrix to match the size, shape, and functional groups of a template molecule, which interacts with the target molecule by making hydrogen bonds and interacting hydrophobically.16−18 The MIPs have attracted considerable research and applications in many fields, such as adsorption,19 online solid phase extraction,20 sensors,21 photocatalysis,22 and isolation.23 In the majority of MIPs, because of noncovalent (especially hydrogen bonding) interaction between the functional monomer and template, molecular recognition occurs in nonpolar or less polar proper solvents. The highest selectivity and rebinding ability toward the template happened while dissolving the analyte in the solvent used for the preparation of MIPs24,25 due to the least swelling of the polymer occurring when exposed to its porogen solvent.26 Therefore, most small organic molecules do not show specific binding in aqueous sample solution, which causes the decrease in MIPs efficiency.27 It is of considerable interest to address this issue by various water-compatible synthesis strategies, such as the preparation of MIPs in water−methanol containing systems, 28 RAFT precipitation polymerization using hydrophilic macromolecular chain-transfer agents,29,30 stimuli-responsive MIPs,31 and imprinting in metal−organic gels such as porogen.32 Our literature survey on the construction of some MIPs for monitoring HCT33,34 reveals some limitations. For instance, they are not based on a water-compatible process. On the other hand, organic solvents are required, such as porogen. In the past decade, synthesis of paramagnetic nanoparticles has been intensively developed for many applications: catalysis,35 drug delivery,36 magnetic separation,37,38 and aqueous applications.39 A number of reports using core−shell magnetic MIPs for recognization, detection, and removal of organic compounds have been demonstrated. Magnetic nanoscaled adsorbents could be easily separated from sample solutions by the employment of magnetic processes. So many papers reported imprinted coatings on a magnetic component.40−43 Core−shell structures of MIPs coatings based on magnetic Fe3O4 NPs have been synthesized via different techniques for recognition and enrichment of various analytes. Ma et al.44 synthesized a core−shell magnetic imprinted polymer by coprecipitation and polymerization for the extraction of quercetagetin. Hu et al.45 synthesized magnetic molecularly imprinted polymers using microwave initiated suspension polymerization and used the MIPs to extract and determine ß-agonists in pork and pig liver samples. Chao et al.46 reported imprinted organic-base polymer shells over magnetic particles for selective determination of hydroquinone. All the magnetic MIPs discussed above were prepared using an organic-based polymer as an imprinted layer, which requires adequate porogen under controlled temperature during 8−24 h. However, urine contains water, inorganic salts, and organic compounds including proteins, hormones, and other metabolites from the body. The biological components, such as proteins and lipids, which are robustly adsorbed on the hydrophobic MIP surfaces, unconstructively interfere with the recognition properties.47 The sol−gel route satisfactorily results in hydrophilic inorganic-based imprinted materials.48 In comparison with acrylic-based MIPs, organically modified sol−gel has been confirmed to be more particular to the target variety, allowing the analytes to diffuse faster in aqueous or alcoholic solutions at mild reaction temperatures.49,50
Herein, the aims of this study follow are as follows: (1) Onepot and green synthesis of Fe3O4@MIPs at room temperature without using any organic solvent, which allows all reagents to react together at desired conditions. This thus makes the experimental period short and reduces labor effort.51 (2) Investigation of the basic characteristics of Fe3O4@MIPs by various instrumental analyses, such as SEM, TEM, FT-IR, and VSM. (3) Application of the Fe3O4@MIPs as SPE adsorbents for the purification and extraction of hydrochlorothiazide from urine samples.
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EXPERIMENTAL SECTION
Materials and Methods. Iron(III) chloride hexahydrate (FeCl3· 6H2O), iron(II) chloride tetrahydrate (FeCl2·4H2O), acetic acid, tetraethyl orthosilicate (TEOS), 3-aminopropyl trimethoxysilane (APTMS), ammonium hydroxide (NH4OH), poly(ethylene glycol) (PEG) with average W% of 6000 g mol−1, and cetyltrimethylammonium bromide (C19TAB) were purchased from Merck (Darmstadt, Germany). HCT powder was purchased from the Ministry of Health and Medical Education (Tehran, Iran). Ultrapure water was prepared from a Milli-Q gradient water purification system. Methanol, ethanol, acetone, and acetonitrile (HPLC grade) were prepared by J.T. Baker (Deventer, Holland). Prior to use, the solutions were filtered using ́ 0.45 μm nylon filters from AnálisisVinicos (Tomelloso, Spain). Human urine samples were collected from healthy volunteers and stored at 4 °C before use. The Fe3O4@MIP was characterized by field emission scanning electron microscopy (FE-SEM, Hitachi S4160, Japan), transmission electron microscopy (TEM, Zeiss, Germany), and Fourier transform infrared (FTIR) spectroscopy using KBr (FTIR-8300, Shimadzu). Its magnetic properties were studied using a vibrating sample magnetometer (VSM, LDJ 9600-1, USA). High-performance liquid chromatography analysis was carried out with an Agilent 1100 liquid chromatograph (Wilmington, DE, USA). It was equipped with a micro vacuum degasser (model G1379A), a quaternary pump (model G1311A), a multiple wavelength detector (model G13658:220 nm for HCT), a sample injection valve with a 20 μL sample loop, and a Knauer C18 column (4.6 mm i.d. 250 mm, 5 μm). Data acquisition and analysis were performed using the Agilent Chemstation software. Water (pH adjusted at 3.0 by phosphoric acid)−methanol (80/20, v/ v) was used as the mobile phase while setting the flow rate to be 1.0 mL min−1. A 0.45 μm filter was used for the filtration of the mobile phase followed by its vacuum-assisted degassing before use. The ambient temperature was chosen to be the condition for the operating system. Synthesis of Fe3O4 Magnetic Nanoparticles. The magnetite nanoparticles were synthesized by the coprecipitation of FeCl2/FeCl3: 15 mmol of FeCl3·6H2O and 10 mmol of FeCl2·4H2O were dissolved in 80 mL of deoxygenated water in a 250 mL trineck flask. 50 mL of ammonium hydroxide solution (28%, W%) was added dropwise to the solution at 300 rpm under nitrogen gas flow to get a black mixture. Then, the obtained black mixture was strongly stirred for 30 min at 80 °C. After the completion of reaction, a magnet was applied to collect the black precipitates. Next, it was repeatedly washed with deionized water until achieving a neutral pH for the washed solution. Preparation of Fe3O4@MIPs (MMIPs) and Fe3O4@NIPs (MNIPs). The surface of Fe3O4 nanoparticles was modified by mixing 2.0 g of the nanoparticles with 10.0 g of PEG in 50.0 mL of deionized water by stirring for 20.0 min. After sonicating for 30 min, 0.01 g of CTAB was added to the mixture to obtain a homogeneously dispersed solution. Then 0.68 mL of ammonium hydroxide solution (25%,w:w) was added drop by drop, followed by adding 0.52 mmol of HCT as a template. After that, 0.55 mL of TEOS and 0.11 mL of APTMS were added where the reaction was performed assisted by stirring at room temperature for 24 h until the completion of hydrolysis. The obtained product was collected using a magnet followed by redispersing in deionized water/ammonium hydroxide (9:1 v/v) solution by applying several times sonication for the removal of templates. Finally, it was B
DOI: 10.1021/acssuschemeng.6b02615 ACS Sustainable Chem. Eng. XXXX, XXX, XXX−XXX
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Figure 1. Preparation of Fe3O4@MIPs procedure.
Figure 2. FT-IR study of (a) Fe3O4, (b) Fe3O4@PEG, and (c) Fe3O4@PEG@MIPs. d-SPE Procedure of HCT from Urine. Under optimized
repeatedly washed with deionized water and dried, assisted by evacuation, and the product was referred as MMIPs. The MNIPs were similarly synthesized except that no template was used. Adsorption Experiments. The evaluation of adsorption was performed to estimate the capacity of MMIPs/MNIPs for recognizing and binding HCT in aqueous solutions. Typically, 50.0 mg of MMIPs or MNIPs was individually suspended in 10.0 mL of HCT solutions over the concentration range 0.05−1.5 mg mL−1 in a glass tube. The samples were shaken in an incubator for 12 h at room temperature followed by their magnetic separation and analysis by HPLC. The difference between the amount of initially added and free HCT was calculated to determine the amount of HCT bound to the MMIPs or MNIPs. All data were collected and processed based on the Freundlich isotherm (FI) to obtain the parameters corresponding to the HCT binding to the MMIPs and MNIPs.
conditions, 10.0 mL of urine sample was placed in 25 mL flasks, and the pH was adjusted to 4.0. Then 50.0 mg of MMIP nanoparticles, previously rinsed with deionized water (pH 4.0), were mixed with urine and sonicated at room temperature for 25 min. The MMIPs were magnetically separated from the solution. Then it was washed with 3.0 mL of deionized water under ultrasound irradiation and eluted with 2.0 mL of ethanol:acetone:ammonium hydroxide (45:45:10) solution assisted by sonication. The eluted solution was dried under nitrogen at room temperature. Finally, it was dissolved in 0.2 mL of mobile phase for analyzing by HPLC-UV. C
DOI: 10.1021/acssuschemeng.6b02615 ACS Sustainable Chem. Eng. XXXX, XXX, XXX−XXX
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Figure 4. Magnetization curve of Fe3O4 (purple line), MMIPs (green), and MNIPs (pink).
Figure 3. SEM images of MMIPs (a) and MNIPs (b), and TEM image of MMIPs (c).
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Figure 5. Adsorption isotherm of MMIPs and MNIPs well fitted to the Freundlich isotherm.
RESULTS AND DISCUSSION Characterization of MMIPs. The schematic procedure of the preparation of MMIP nanoparticles is shown in Figure 1.52 At the beginning, Fe3O4 nanoparticles were prepared by a coprecipitation method. Although magnetic nanoparticles synthesized using a solvothermal method are of magnetic response higher than those prepared by the coprecipitation method,53 we used the coprecipitation method due to no organic solvent consumption in this method. The protonated F3O4can electrostatically interact with the hydroxyl groups of PEG. In addition, the hydroxyl groups of PEG can form a hydrogen bond with the hydroxyl groups of magnetite nanoparticles in aqueous solution. Therefore, PEG could encapsulate Fe3O4 nanoparticles. After that, a sol−gel reaction was employed for hydrolysis of the silanes of TEOS and
APTMS to achieve molecularly imprinted particles anchored by hydrogen bonding to PEG. Due to the presence of several functional groups, HCT can simply react with the amine group of APTMS via a hydrogen bond; thus, APTMS was chosen as a functional monomer. Subsequently, molecularly imprinted silica particles initially interact with CTAB micelles, causing more aggregation via the hydrogen bonding of PEG. Ammonium hydroxide was used as both a catalyst and suitable basic solution for dissolving HCT. Finally, molecularly imprinted particles embedded on/in the surface of magnetic nanoparticles were obtained at green and mild conditions. It is worth noting that the properties of the hydrophilic sol−gel make Fe3O4@MIPs excellently dispersible in water. D
DOI: 10.1021/acssuschemeng.6b02615 ACS Sustainable Chem. Eng. XXXX, XXX, XXX−XXX
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Figure 6. Recoveries of HCT and analogues in single-solution onto MMIPs (a) and MNIPs (b), and in mixed-solution onto MMIPs (c).
Table 1. Linearity, Precision, and Accuracy of the MMIPs-HPLC/UV and MNIPs-HPLC/UV Methods MNIPs
MMIPs
Recovery (%)
Recovery (%)
Interday (n = 4)-RSD (%) 48.5−3.9 59.5−4.4 58.9−3.2
Intraday (n = 4)-RSD (%)
Interday (n = 4)-RSD (%)
Intraday (n = 4)-RSD (%)
Add (μg L−1)
45.5−2.8 52.0−2.7 61.0−2.3
103.0−6.6 110.0−3.7 93.0−2.6 97.5−1.3
109.5−6.6 90.7−3.3 94.7−3.6 101.0−0.8
2.5 10 100 500
Linear range (μg L−1)
Samples
2.5−1000
Urine
O asymmetric and symmetric stretching vibrations, respectively. The characteristic peak at 3435 cm−1 for N−H stretching was ascribed to APTMS. The absorbance peaks of C−N and C−H were observed at 1635 and 1385 cm−1, respectively.54 These results indicate that amine groups present in the sol−gel make hydrogen bonds with HCT on the surface of magnetic nanoparticles.
FT-IR spectra of MMIPs are shown in Figure 2. The absorption band of Fe−O at 579 cm−1 was found in MMIPs, demonstrating that Fe3O4 was embedded in these materials. As sol−gels have a high water adsorbing capacity and Fe3O4 anchored by molecularly silica particles, a broad absorption band was observed at 3300 cm−1, which is attributed to O−H stretching of adsorbed water on Fe3O4@MIPs nanoparticles. The typical bands at 1072 and 800 cm−1 correspond to the Si− E
DOI: 10.1021/acssuschemeng.6b02615 ACS Sustainable Chem. Eng. XXXX, XXX, XXX−XXX
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high adsorption capacity and eliminating the leaching of template during extraction and analytical errors. As seen from the TEM image of MMIPs shown in Figure 3(c), there are three regions of different contrasts which support the schematic picture (Figure 1) of the prepared MMIPs and suggest that the silica MIP particles were deposited onto the PEG-CTAB brushes. The resulting MMIP nanoparticles consist of Fe3O4 nanoparticles of average size of 30 nm (darkest in TEM image), silica nanoparticles (mid-dark), and PEG-CTAB brushes (less dark). Lin and co-workers55 reported the synthesis of superparamagnetic-MIP with magnetic saturation at 49.7 emu g−1. Although these superparamagnetic MIPs had high saturation magnetization, their method required several steps: toxic organic solvent (cyclohexane), high temperature, and time wasting. The magnetization curve obtained from a vibrating sample magnetometer (VSM) shows that the magnetic saturations of Fe3O4, MMIPs, and MNIPs were 61.0, 51.0, and 50.5 emu g−1, respectively (Figure 4). In addition, the zero coercivity of the prepared nanoparticles confirms their superparamagnetic properties. The high saturation magnetization of such prepared MMIPs causes high susceptibility to external magnetic fields, which makes them easily and quickly separable from sample solution, as well as the magnetic saturation of MMIPs being similar to that of MNIPs, which corresponds to the same polymerization for both MMIPs and MNIPs. As shown in Figure S1, MMIPs are highly homogeneously dispersed in a urine sample where an external magnetic field is exerted. After applying the external magnetic field, all MIPs are quickly attracted to the vial wall. Binding Isotherms. The adsorption isotherms of HCT in the MMIPs and MNIPs are shown in Figure 5. As is shown, an increase in the equilibrium adsorption capacity is observed while increasing the initial concentration of HCT. The imprinted cavities become saturated while reaching the concentration of 1.2 mg mL−1. The adsorption data can be fitted using the FI model below.56
Figure 7. Reusability of MMIPs. Spiked concentration 200.0 μg L−1.
Log Q = m Log C + Log α
(1)
where Q and C are the amount of analyte adsorbed per unit mass of polymer at the equilibrium analyte concentration on adsorbent and the analyte concentration in solution, respectively. The parameter α is the Freundlich constant, and m is the heterogeneity factor. The parameter m can take values from 1 to 0, where the values of 1 and 0 indicate the homogeneity and heterogeneity. As seen in Figure 5, both prepared polymers heterogeneously contain binding sites while the m value of MMIPs is lower than that of MNIPs, which is due to the higher amount of high-affinity binding sites in the
Figure 8. Chromatograms of the (a) blank and (b) spiked urine samples.
From SEM images, the particle size and morphology of MMIPs and MNIPs were found to be similar (Figures 3(a) and 3(b)). This means that the molecular template does not obviously affect the morphology of prepared polymers. MMIPs are bulk materials that consist of many small spheres which adhered to each other. These uniform structures provide suitable conditions for complete template removal, inducing
Table 2. Determinations of HCT in Two Human Urine by Different Adsorbents MMIPs −1
Sample
Added (μg L )
Urine 1
0.0 10.0 50.0 200.0 0.0 10.0 50.0 200.0
Urine 2
MNIPs
C18
Recovery (%)
RSD (%)- (n = 3)
Recovery (%)
RSD (%)- (n = 3)
Recovery (%)
RSD (%)- (n = 3)
92.7 96.7 103.8
2.2 3.2 1.1
55.0 58.0 64.8
3.1 3.4 1.2
42.3 53.0 60.7
5.9 1.9 1.3
102.0 93.0 96.3
2.6 2.8 1.2
59.0 54.3 66.2
3.9 3.8 1.9
48.3 44.4 59.2
3.2 4.8 1.3
F
DOI: 10.1021/acssuschemeng.6b02615 ACS Sustainable Chem. Eng. XXXX, XXX, XXX−XXX
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Voltammetry HPLC-UV
HPLC-DADe HPLC-MS
Cyclic voltammetry. bMolecularly imprinted solid phase extraction. cLiquid chromatography−tandem mass spectrometry. dNot reported. eHigh performance liquid chromatography−diode array detector. Liquid−liquid extraction.
Lack of selectivity Selective MMIPs-d-SPE
LLEf LLE
Lack of selectivity Lack of selectivity
0.500−200 ng mL−1 25.0−25000 ng mL−1 0.05−20 μg mL−1 1−100 ng mL−1 0.05−20 μg mL−1 3.0 × 10−6− 7.4 × 10−5 M 2.5−1000 μg L−1 Lack of selectivity SPE
Plasma Urine Urine Plasma Urine Tablet Urine
9 × 10 −1 × 10 0.1−21.0 μg mL−1 Selective Selective MISPEb Tablet and serum Tablet and serum
CV UV−vis Spectrometry LC-MSc
a
MMIPs structure. These results demonstrate the important role of the template molecule in the heterogeneity of the MMIPs during the imprinting process. The adsorption capacity of MMIPs was 84.34 mg g−1, 3.0 times more than that on MNIPs (28.10 mg g−1), which is due to imprinted specific cavities in the MMIPs structure. MNIPs were prepared identically to the MMIPs except that the templates were not added during synthesis. The functional monomer (APTMS) in the MNIPs could react with HCT through a hydrogen bond, so MNIPs may also seize some HCT molecules. Selectivity Evaluation. For the evaluation of MMIPs selectivity, the equilibrium adsorption capacity of HCT, or one of the three compounds, of similar structure, including chlortalidone, bendroflumethiazide, and diazoxide, was calculated (Figure S2). For sampling, 50.0 mg of MMIPs was added into a glass tube containing 10.0 mL of methanol solution consisting of the species studied at concentrations of 0.5 and 1 mg mL−1. Next, these solutions were kept under stirring at room temperature for 24 h. Subsequently, the particles were magnetically collected, followed by individual monitoring of the nonaccumulated contents of components in supernatant solutions using the calibration curve constructed under the same conditions. As shown in Figure 6a, MMIPs had the highest affinity for HCT compared to other compounds. The adsorption capacity of MMIPs for chlortalidone was better than others, which probably is related to the resemblance in functional groups, which could generate hydrogen bonds as in HCT. For bendroflumethiazide, the presence of several substitutions increased the volume of the molecule, which caused an increase in steric hindrance to enter the MMIPs cavity and decrease affinity. The adsorption capacity for diazoxide was better than that of bendroflumethiazide, which could be explained by the similar molecular volumes of diazoxide compared to HCT that increase the opportunity for diazoxide to enter the MMIPs cavity. Although diazoxide is similar in terms of volume of HCT, there was a large distinction in the functional groups position. Thus, the selectivity pattern indicated the significant contribution of the size and functional groups to the selectivity. In addition, the specificity of the MMIPs for their competitive adsorption in quaternary mixtures was investigated (Figure 6c), which revealed a decrease in the capacity of sorbent toward each of four compounds in comparison to their individual value, while the extent of signal reduction for HCT was lower than that of other components. The results confirm that MMIPs possess special recognition sites and prove the high applicability of the applied method with high selectivity for the determination of HCT. Optimization of d-SPE Parameters in Urine. By the adoption of MMIPs as sorbents, several factors influencing the extraction and desorption process, including pH, adsorbent mass, extraction sonication time, and desorption sonication time, were evaluated and optimized by extracting HCT spiked blank urine at the level of 200.0 μg L−1. For the full explanation of the optimization approach, the type of washing and desorbing solvent was preliminarily investigated using an univariate method. Washing Condition. The washing solvent should elute accumulated matrix interferences as much as possible in the least volume. In this study, based on the fundamental green chemistry, to keep away from halogenated solvents, several washing solvents with different polarities, including methanol, deionized water, acetonitrile, and hexane, were examined in order to find the most proper solvent for washing. It was found
f
5.5 ng mL−1 NR NR 1.2 × 10−6 M 0.75 μg L−1
NRd
M and 1 × 10 −1 × 10
−2 −5 −5 −10
Linear range Sample pretreatment selectivity Sample Pretreatment Sample Method
Table 3. Comparison of Different Methods with the Proposed Method for the Determination of HCT
a
61 Present work
59 60
