Efficient removal of digoxin from aqueous solution using magnetic nanocomposite (Fe3O4–GO–SO3H) as an advanced nano-absorbent

Abstract Digoxin separation from pharmaceuticals wastes, is small piece of the larger puzzle in holistic risk assessment. In this study, a novel magnetic nano composite (graphene oxide/Fe3O4/SO3H) was synthesized and used as an absorbent for the removal of digoxin from aqueous solution. We utilized UV-Vis spectrophotometry (UV/Vis) for detection and efficient removal of digoxin by magnetic graphene oxide (MGO) in different concentrations. Magnetic absorbent was characterized by thermal gravimetric analysis (TGA), Fourier-transform infrared spectroscopy (FTIR) and X-ray diffraction (XRD). The optimized concentration of absorbent and digoxin were 500 and 1 ppm respectively, in which the optimize reaction time was lasting 10 min. Finally, under optimized condition, MGO was used for the efficient separation of digoxin from aqueous solution. GRAPHICAL ABSTRACT


Introduction
Digoxin is a glycosylated steroid-like drug which is extracted from foxglove for the first time in 1930.
health care system. This drug is essential to cure various heart conditions such as atrial fibrillation, atrial flutter, and heart failure [2,3]. This cardiac glycoside drug is the fifth most commonly prescribed drug in US and costed less than 25 USD per month in 2015 [4]. Ubiquitously, this drug is prescribed in congestive heart failure that inhibits the Na þ /K þ ATPase pump and enhances the intracellular concentration of cytosolic calcium, subsequently. The intracellular Ca þ2 , then adjusts various physiological events such as cardiac muscle contraction [5,6]. Beside the importance of digoxin as a widely prescribed drug, the toxic effects of this drug both in environment and medicine cannot be neglected. Wide variety of environmental samples including sewages, ground waters and drinking waters are at risk of pharmaceuticals wastes contamination [7]. Aquatic pollution is particularly troublesome since it affects life-cycle of aquatic organisms, plants and drinking water [8]. Even, very low amount of this wastes is worrisome because their affects accumulates and leading to irreversible changes over the years. Therefore, there is a significant need for detection, separating, and removing them from wastes.
Digoxin separation from pharmaceuticals wastes, is small piece of the larger puzzle in holistic risk assessment. Various surveys have been conducted to filtration and refinement of sewages. Moreover, multiple techniques have been introduced for detection of digoxin in human serum samples such as liquid chromatography (HPLC) [9], immunochemical assays [10] and biosensors [11]. However, there is an urgent need for specific, precise, simple and cost effective method for separation of digoxin from aqueous environments.
Graphene is two-dimensional carbon nanostructures which consisting mechanical, thermal and electrical characteristics [12]. The oxidation product of graphene is graphene oxide (GO), which possesses hydroxyl, epoxide, carboxyl and carbonyl functional groups [13][14][15][16][17][18]. These functionalized groups not only supply plentiful active attachment sites but also provide excellent adsorption sites for abundant contaminants in aqueous solution [19]. Therefore, multiple functionalised magnetic absorbents has been synthesised and introduced by researchers in order to adsorb and remove various compounds such as proteins [20], metal ions [21] and drugs [22]. Several pharmaceutical companies produce remarkable amounts of toxic pharmacy and their derivatives which are the most important hazard compounds in wastewater. Abundant studies have been conducted to eradicate compounds from environment by taking advantage of organic absorbents [23] such as separation SO 2 , NO [24], CO 2 [25], methylene blue [26], Ca, and Mg [27] from aqueous solution. However, fewer researchers have been conducted for pharmacy separation from aqueous solution.
Due to the high digoxin toxicity, there is an urgent need for the separation of this drug from the wastes. Multiple techniques have been introduced for digoxin monitoring in biological samples such as liquid chromatography (HPLC) [27], LC-MS/MS assays [28], immunochemical assays [29], and sensing [30]. Most of the methods for digoxin determination are time-consuming, expensive and complex. Moreover, they requires sophisticated experts for sample preparation and cause harmful effects to the environment. Therefore, there is a considerable attention to introduce rapid, sensitive and greener method for digoxin detection and eradicating [31].
The goal of this study is to expand an analytical procedure exploiting for the first time the precise extraction based on a novel absorbent aiming at the spectrophotometric determination of digoxin. The procedure was based on formation of magnetic nano composite (Fe 3 O 4 -GO-SO 3 H) and its interaction with digoxin in aqueous solution.
UV-spectrophotometric method enables quantitative measurement of the reflection or transmission properties of digoxin and reveals how much a chemical substance absorbs light by evaluating the intensity of light as a beam of light passes through digoxin solution. This method offers cost effective and time saving alternative to other analytical methods and is more specific than electromagnetic spectroscopy, near ultraviolet, and near infrared techniques [22,23].
In this study, Fe 3 O 4 -GO-SO 3 H was synthesized and used as a magnetic absorbent for the removal of digoxin from aqueous solution. Then, the peak intensity of UV/vis absorbent was measured in the presence and absence of digoxin by UV-spectrophotometric.
Therefore, simple and fast procedure introduced for digoxin detection and eradication from aqueous solution.

UV-1800
UV-VIS Spectrophotometer from Shimadzu, Labnet's Vortex Mixer VX-200 and New Brunswick Innova 4000 Incubator Shaker were used in this study. The centrifugation was performed on a KUBOTA 6800 centrifuge (KUBOTA Corporation, Japan). X-ray diffraction (XRD) patterns of materials were recorded on a Siemens D 5000 X-Ray diffractometer (Texas, USA) with a Cu K a anode (k ¼ 1.54 A Å) operating at 40 kV and 30 mA. TEM analysis was conducted on a Carl Zeiss LEO 906 electron microscope operated at 100 kV (Oberkochen, Germany). Fourier transform infrared (FTIR) spectra were measured using by a Shimadzu model FTIR prestige 21 spectrophotometer (Tokyo, Japan) using KBr discs. The surface morphology of the synthesized adsorbents were evaluated with a FESEM analysis, which was conducted on TESCAN system of FEG-SEM MIRA3 TESCAN (Brno, Czech Republic).

Synthesis of graphene oxide (GO)
Hummer method was utilized to prepare GO from purified natural graphite [32] According to modified Hummer method, 0.5 g of graphite powder was added to 50 mL of 98% H 2 SO 4 in an ice bath. Furthermore, in adjusted temperature, shaking solation was mixed with 2 g of KMnO 4 slowly and stirred for 2 h at temperatures below 10 C and followed by 1 h shaking at 35 C. So, 50 mL of DI water was used to dilute reaction mixture in temperature below 100 C. After 1 h stirring DI water utilized to diluted solution to approximately 150 mL. Further, in order to change the color of solation to brilliant yellow 10 mL of 30% H 2 O 2 was used. After plenty washing processes the pH of the supernatant become neutral. Finally the resulting solid was dried and brown powder was obtained.

Synthesis of Fe 3 O 4 -GO-SO 3 H (magnetic adsorbent)
In order to synthesis of Fe 3 O 4 -GO-SO 3 H graphene oxide was mixed with FeCl 3 .6H 2 O and FeCl 2 .4H 2 O (2:1 mole ratio). Then this solation was ultrasonicated for 30 min, and 20 mL of 30% ammonia solution in DI water was added to above solution.
Eventually brown powder of Fe 3 O 4 doped GO is ready to use [33, 34].

Characterization of absorbent (Fe 3 O 4 -GO-SO 3 H) before adsorption of drug
SEM and TEM microscopy were occurred to reveal morphological characteristics of Fe 3 O 4 -GO-SO 3 H. (Figure 1(A,B)) single atomic layer, thickness nano structures and their dispersity were observed. FESEM analysis confirmed the presence of Fe 3 O 4 on the surface of GO. According to TEM images presence of Fe 3 O 4 nanoparticles on GO structure was appeared as bright dots (Figure 1(B)). Moreover, spherical shape and the distribution of Another important factor that affects the application of absorbent is thermal stability. Thermal stability of Fe 3 O 4 -GO-SO 3 H was investigated by TGA in dry air ( Figure 4). The mass loss (64%) might mainly result from the oxidation of carbon and removal of oxygen containing groups. The distinct mass loss appeared at the temperature higher than 170, indicating as-prepared Fe 3 O 4 -GO-SO 3 H has a good thermal stability and can be used as a suitable absorbent in wastewater treatment (Figure 4).

Characterization of Fe 3 O 4 -GO-SO 3 H after adsorption of digoxin
XRD and FT-IR was conducted to prove structural properties of Fe 3 O 4 -GO-SO 3 H (Figures 2 and 3). Another important factor that affects the application of adsorbent is its thermal stability. The FTIR spectra of the Fe 3 O 4 -GO-SO 3 H and digoxin/Fe 3 O 4 -GO-SO 3 H, were shown in Figure 5. The FTIR spectrum of absorbent showed a peak between 500 and 750 cm À1 (Fe-O), verifying the existence of Fe 3 O 4 , and they both presented the peaks around 1609 cm À1 (C ¼ O stretching vibrations of -COOH) and 3420 cm À1 (-OH), which confirmed the presence of GO. Moreover, according to previous studies conducted by previous researchers, bare GO reveals plenty characteristic bands at 1719, 1222, and 1060 cm À1 , which are ascribed to the GO carbonyl stretching, O-H deformation vibration, and C-OH and C-O stretching, respectively. The infrared spectrum of pure digoxin revealed absorption bands at $3400 cm À1 related to -OH stretching vibration; at $3000-2900 cm À1 equivalent to C ¼ CH 2 and CH 2 stretching vibration; at $1750 cm À1 corresponding to -C ¼ O stretching vibration; at $1400 cm À1 attributed to conjugated C ¼ C of aromatic groups; at $1300 cm-1 equivalent to the bending mode of -CH3 and at $1200-1000 cm À1 corresponding to skeletal aromatic ring vibration. The FTIR results reported for pure digoxin were similar to those previously described.
The XRD spectra were recorded in a range of 2h from 5 to 50 and show (001) diffraction peak at 2h ¼ 11.0 for GO, indicating the distance between graphene layers. The XRD pattern of MGO is very similar to that of the pristine Fe 3 O 4 , with diffraction peaks which can be indexed as the characteristic

Adsorption study
All experiments were performed by providing 500 ppm of Fe 3 O 4 -GO-SO 3 H as absorbent. Then different concentrations of aqueous solution of absorbent (10-20-40-80-100-500 ppm) was provided from main stock by DI water. In order to carry out the absorption process, the pre-prepared digoxin solutions was incubated with absorbent for 1 h. In the following step, incubated solutions were centrifuged for about 15 min in 1000 rpm. Finally, deposited of adsorption sediment was collected. Then, the UV/Vis spectra of digoxin and absorbent was recorded at various concentrations ( Figures 5 and  6). So, the absorbent concentration, amount of digoxin and incubation time were optimized as 500 ppm and 1 ppm at 10 min, respectively ( Figure  7). Finally, deposited of adsorption sediment was collected.

Optimization of digoxin and absorbent concentration and reaction time
In order to optimize the reaction condition occurred between digoxin and absorbent, a multiple concentrations of absorbent (10-20-40-80-100 ppm) and digoxin (0.5, 1, 5, 10, ppm) was made. Successive, reactions were performed with different concentrations of digoxin at different incubation times. The optimize saturation point of absorbent concentration in which absorbent is saturated was estimated as 500 ppm. Furthermore, the minimum amount of digoxin that absorbed was estimated as 1 ppm (Figures 5 and 6). Incubation time was the other important factor which was optimized in 10 min (Figure 7).

Optimization of digoxin and absorbance concentration and reaction time
In order to optimize the reaction condition occurred between digoxin and absorbent, multiple concentrations of absorbent (10-20-40-80-100 ppm) and verity digoxin solutions (0.5, 1, 5, 10, ppm) was mixed. Successive, reactions were performed with different concentrations of digoxin at different times. The optimize saturation point of absorbent concentration in which absorbent is saturated was estimated 500 ppm. Furthermore, the minimum amount of digoxin that absorbed was estimated 1 ppm. Incubation time was the other important factor which were optimized in 10 min. UV-Vis spectrophotometry was used in this work for determination absorbance of digoxin by  Adsorption is often regarded as a common method for the removal of pollutants in gas and liquid phases. Several strong points of this approach include cost-effectiveness and high performance. Thanks to the good reusability of absorbents, the overall cost can be reduced. Moreover, the adsorption is highly compatible with organic-derived hazardous wastes including digoxin contaminant. Therefore, the IBU degradation by the adsorption process has been rapidly developed. According to the above discussed example, in the adsorption process, solid adsorbents play a decisive role in removing the contaminants. Heterogeneous materials have undergone a long history, especially nanostructured graphene based magnetic nanomaterials. Thanks to the high surface area along with open metal sites, these materials have been considered as ideal platforms towards removal of drug residues.
Comparison of the performance of Fe 3 O 4 -GO-SO 3 H with previous reports indicated that, proposed adsorbed has various advantage such as magnetic properties, high surface are and excellent electrostatic interaction which lead to efficient removal of digoxin from aqueous solution. Du to existence of Fe 3 O 4 NPs as magnetic core, this adsorbent can be separated using simple external magnetic and there is no need to high cost method for the removal of adsorbent from sample solutions. Also, high surface area of GO lead to dense loading of digoxin on the surface of nanocomposite (adsorbent) and high yield of reaction was obtained. On the other hand acidic agent on the structure of adsorbent is important item which lead to electrostatic interaction with the candidate contamination (digoxin). Therefore, purposed adsorbent is an excellent candidate than other materials for this purpose. According to the magnetic studies a-Fe 3 O 4 -GO-SO 3 H, nanocomposites exhibit a ferromagnetic behavior with small coercivity and remnant magnetization at room temperature, which is desirable for many practical applications, such as water purification systems, as it can be removed from the contaminated water. The hybrid material was separated after being exposed to the magnetic field. However, additional studies should be done since a-Fe 2 O 3 nanoparticles reduce the adsorption capability of GO-SO 3 H. The magnetization is probably enough to allow magnetic separation in laboratory-scale systems but might not be high enough to allow separation in large wastewater volumes.

Conclusion
In this study, Fe 3 O 4 -GO-SO 3 H was synthesized and utilized to absorption and removal of digoxin from aqueous solution. Fe 3 O 4 -GO-SO 3 H (adsorbent) was characterized by Scanning electron microscopy (SEM), Transmission electron microscope (TEM), thermal gravimetric analysis (TGA), Fouriertransform infrared spectroscopy (FTIR) and X-ray diffraction (XRD). All characterization analyses confirmed the absorption ability of Fe 3 O 4 -GO-SO 3 H and separation of digoxin by candidate absorbent. The optimized concentration of absorbent and digoxin were 500 and 1 ppm respectively, in which the optimize reaction time was lasting 10 min. Therefore, this simple, cost effective and reliable method for the separation of digoxin from aqueous solution, could pave a new way for solving small piece of the larger puzzle in holistic risk assessment.

Acknowledgments
We thank Tabriz University of Medical Sciences for instrumental supporting of this research.

Disclosure statement
No potential conflict of interest was reported by the authors.

Notes on contributors
Paria Pashazadeh-Panahi received his M.Sc. in biochemistry from Gholestan University of Medical Science. She is working mainly on synthesize of nanomaterials and its appplication on healthcare science. Also, She is an resercher in the field of biotechnology. She is a researcher with over 12 publications and research interests in optical sensing and biomedical analysis.