TY - JOUR
AU - Shekarchi,, Maryam
AB - Abstract In the present study, a facile modified impregnation method was employed to synthesize superparamagnetic graphene oxide–Fe3O4 (GO–Fe3O4) nanocomposites. Based on the GO–Fe3O4 as adsorbent, a simple and fast magnetic-dispersive solid phase extraction followed by high performance liquid chromatography with fluorescence detection (M-dSPE–HPLC–FL) method was established and validated for the preconcentration and determination of terazosin hydrochloride (TRZ) in human plasma samples. The obtained nanomaterials were characterized by X-ray diffraction, Fourier transform infrared spectroscopy, scanning electron microscopy, energy dispersive X-ray spectroscopy and vibrating sample magnetometry. Different parameters affecting the extraction efficiency, such as sample pH, amount of sorbent, extraction time, elution solvent and its volume and desorption time, were evaluated and optimized. The linearity of the proposed method was excellent over the range 0.3–50.0 ng mL−1 with an acceptable coefficient of determination (R2 = 0.9989). The limit of quantification and limit of detection were found to be 0.3 and 0.09 ng mL−1, respectively, and the preconcentration factor of 10 was achieved. Intra- and inter-day precision expressed as relative standard deviation (RSD %, n = 6) were between 2.2–3.8% and 4.7–6.4%, respectively. Accuracy, estimated by recovery assays, was 97.7–106.6% with RSD ≤ 5.2%. Ultimately, the applicability of the method was successfully confirmed by the extraction and determination of TRZ in human plasma samples. Introduction Terazosin hydrochloride (TRZ; Figure 1), 1-(4-amino-6,7-dimethoxy-2-quinazolinyl)-4-[(tetrahydro-2-furanyl)carbonyl]-piperazine monohydrochloride, an alpha-1 adrenergic receptor antagonist, is classified as a quinazoline-derivative with a long-lasting action (1). TRZ is used to treat the symptoms of benign prostatic hyperplasia (BPH) and for the treatment of lower urinary tract (LUT) problems by relaxing the muscles of the prostate and bladder. It is also used in high blood pressure (hypertension) by relaxing veins and arteries (2). TRZ is rapidly absorbed from the gastro-intestinal tract after oral administration (90% bioavailability) and is extensively metabolized by the liver (3). TRZ is widely used for the treatment of BPH/LUT symptoms. Therefore, it is important and necessary to develop reliable bioanalytical method for its detection in biological samples. Literature survey revealed that various analytical techniques such as chromatographic, spectrophotometric and electroanalytical methods have been reported for the determination of TRZ in various matrices (4). Many of these methods are, however, relatively expensive, not sensitive and unselective and not accurately reliable for the quantitation of trace amounts of TRZ. Among these methods, spectrofluorimetry (5) and high performance liquid chromatography (HPLC) with ultraviolet (UV; 6, 7), fluorescence (FL; 8, 9) and mass spectrometric (MS) detection (10) are available for TRZ determination in biological fluids. The expensive equipment required for LC–MS methods are not available in most of laboratories. However, compared to these methods, HPLC–FL detection is still broadly used due to its lower cost and greater robustness in bioequivalence studies. A sample clean-up procedure is one of the essential steps in the whole bioanalytical process, due to trace concentrations of analytes, endogenous interference, complex matrix and small volume of biological samples. Conventional sample preparation methods include liquid–liquid extraction (LLE) and solid phase extraction (SPE). However, LLE has some limitations, such as time-consuming extraction steps, loss of target analytes and needs large amounts of organic solvents that are usually expensive and toxic. SPE typically requires a reduced amount of organic solvents relative to LLE, but the isolation and the enrichment of individual compounds can be laborious. In addition, it is tedious, time-consuming and relatively costly due to expensive cartridges (11, 12). In order to overcome these shortcomings, Anastassiades et al. (13) introduced a simple, fast, inexpensive and effective sample pretreatment technique named dispersive SPE (dSPE). The dSPE reduces the consumption of organic solvent and has wide applicability to various types of analytes and samples. In this technique, a much smaller amount of SPE adsorbent is dispersed in the whole sample solution containing the target analytes instead of being packed into a SPE cartridge, and the adsorbent is then isolated from the extract solution by centrifugation and eluted by an appropriate solvent to desorb the analytes (14, 15). In conventional SPE or SPE-derived techniques, the choice of suitable adsorbent is an essential factor to obtain high preconcentration factor and good recovery, while variety of sorbents can be employed with dSPE. Carbon-based nanomaterials such as carbon nanofibers (16), carbon nanotubes (CNTs; 17) and graphene (18) are exploited extensively due to their large specific surface area, high chemical and mechanical stability. In the case of CNTs, the inner walls are not responsible for the adsorption, due to steric hindrance (19). Graphene, a two-dimensional structure with carbon atoms arranged in a hexagonal (honeycomb) lattice, has excellent physicochemical properties such as large surface area, notable biocompatibility and superior electron mobility. Recently, graphene oxide (GO), the oxidized derivative of graphene, has captured the interests of many researchers as an extraction approach for the determination of trace analytes in various matrices (20–22). GO, in contrast, contains much more polar moieties on the surface, such as hydroxyl, epoxy, carbonyl and carboxyl groups, and thus has a more polar and hydrophilic character than graphene (19, 23). Due to the presence of these functional groups, GO can easily disperse in water, organic solvents, and different matrices but cannot be easily isolated from aqueous solution by traditional separation techniques (e.g., filtration and/or centrifugation). To solve these problems, coating Fe3O4 magnetic nanoparticles (MNPs) onto GO to fabricate superparamagnetic GO nanocomposite is a superior choice, which can ensure the fast, convenient and efficient magnetic separation after adsorption or regeneration. Fe3O4 MNPs are biocompatible, non-toxic and superparamagnetic at room temperature and possess large specific surface area due to their nano nature (24). In the past few years, magnetic-dSPE (M-dSPE) has been used as a new dSPE method based on GO–Fe3O4 for the extraction and enrichment of trace analytes because of its simplicity of operation, rapidity phase separation, high recovery and enrichment factor and capability of combination with different detection techniques (25–27). However, information is lacking on the determination of trace levels of TRZ by using superparamagnetic GO–Fe3O4 nanocomposites as the sorbent. In the present study, Fe3O4 MNPs and GO nanosheets were synthesized by simple modified Massart and Hummers methods, respectively, and then, the Fe3O4 were successfully coated on the surface GO by a modified impregnation method (28, 29). The prepared nanomaterials were characterized by X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FT-IR), scanning electron microscopy (SEM), energy dispersive X-ray spectroscopy (EDX) and vibrating sample magnetometry (VSM). To evaluate the capability of GO–Fe3O4 nanocomposites as M-dSPE adsorbent, TRZ was selected as target analyte in complex biological matrix. The influences of sorbent amount, pH of sample solution, extraction and desorption conditions were optimized, and based on the superparamagnetic GO–Fe3O4, a simple, low-cost, rapid and robust M-dSPE–HPLC–FL method was established. The developed method was validated for the preconcentration and determination of trace amounts of TRZ in human plasma. Figure 1 Open in new tabDownload slide Chemical structure of TRZ. Figure 1 Open in new tabDownload slide Chemical structure of TRZ. Table I Intra- and Inter-day Precision and Accuracy Data for the Determination of TRZ in Spiked Human Plasma Samples (n = 6) Analyte Spiked value (ng mL−1) Found value (ng mL−1) Accuracy Precision (RSD %) Recovery (%) RSD (%) Intra-day Inter-day TRZ 0.3 0.32 106.6 5.2 3.8 6.4 20.0 19.90 99.5 4.4 2.9 5.9 50.0 48.85 97.7 3.1 2.2 4.7 Analyte Spiked value (ng mL−1) Found value (ng mL−1) Accuracy Precision (RSD %) Recovery (%) RSD (%) Intra-day Inter-day TRZ 0.3 0.32 106.6 5.2 3.8 6.4 20.0 19.90 99.5 4.4 2.9 5.9 50.0 48.85 97.7 3.1 2.2 4.7 Open in new tab Table I Intra- and Inter-day Precision and Accuracy Data for the Determination of TRZ in Spiked Human Plasma Samples (n = 6) Analyte Spiked value (ng mL−1) Found value (ng mL−1) Accuracy Precision (RSD %) Recovery (%) RSD (%) Intra-day Inter-day TRZ 0.3 0.32 106.6 5.2 3.8 6.4 20.0 19.90 99.5 4.4 2.9 5.9 50.0 48.85 97.7 3.1 2.2 4.7 Analyte Spiked value (ng mL−1) Found value (ng mL−1) Accuracy Precision (RSD %) Recovery (%) RSD (%) Intra-day Inter-day TRZ 0.3 0.32 106.6 5.2 3.8 6.4 20.0 19.90 99.5 4.4 2.9 5.9 50.0 48.85 97.7 3.1 2.2 4.7 Open in new tab Experimental Chemicals and reagents Reference standard TRZ (purity 99.8%) and graphite powder (purity > 99%, 325 mesh) were purchased from Sigma-Aldrich (Buchs SG, Switzerland). Ferric chloride hexahydrate (FeCl3·6H2O), ferrous chloride tetrahydrate (FeCl2·4H2O), sodium nitrate (NaNO3), sulfuric acid (H2SO4), aqueous ammonia (NH3–H2O), potassium permanganate (KMnO4), hydrogen peroxide (H2O2) with the highest purity (analytical grade) and all HPLC grade solvents were obtained from Merck Co (Darmstadt, Germany). Water used for dilution of aqueous solutions was prepared by an ultra-pure water purifier system (Elga PURELAB UHQ, UK) at resistance of 18.2 MΩ cm−1. Instruments Characterization of the synthesized nanomaterials (GO, Fe3O4 and GO–Fe3O4) was carried out by using FT-IR, XRD, SEM, EDX, VSM and zeta potential analyzer. FT-IR spectra (400–4000 cm−1) were recorded with a Bomem MB 155S FT-IR Spectrophotometer (Québec, Canada) using the KBr pellets technique. XRD patterns were collected on a PANalytical X’Pert Pro MPD diffractometer (Almelo, the Netherlands) using Cu-Kα radiation (λ = 1.5406 Å) under an accelerating voltage of 40 kV and a current of 30 mA in a range angle (2θ = 4–80°) at 25°C. SEM images and EDX spectra were obtained by a VEGA3-SB TESCAN system (Brno, Czech Republic) at an accelerating voltage of 20 kV and 10 kV, respectively, and all samples were sputter-coated with a very thin gold (Au) layer before the SEM/EDX characterization. The magnetic properties were analyzed using a Lakeshore VSM model 7400 (Westerville, Ohio, USA) with magnetic fields of up to 20 kOe at ambient temperature. Zeta potential measurements were done by a Zetasizer (Nano-ZS, Malvern Instruments Ltd, Malvern, UK), and the GO and Fe3O4 samples were diluted to 1 mg mL−1 before measurement. All pH adjustments were done by using a Metrohm digital 744 pH meter (Metrohm Ltd, Herisau, Switzerland) equipped with a combined glass-calomel electrode. Chromatography apparatus and conditions A Waters HPLC system (Alliance, Milford, USA) equipped with a Waters FL detector, a Waters 717plus auto sampler, a multi solvent gradient Waters pump and a vacuum degasser were used for TRZ determination in human plasma. Empower software system (Waters Corporation) was utilized for chromatographic data collection and processing. The chromatographic separation was performed on an ACE5-C18 (250 mm × 4.6 mm I.D., 5 μm particle size) column (Advance Chromatography Technologies Ltd, Aberdeen, UK) at 25°C. The isocratic mobile phase consisted of acetonitrile:phosphate buffer solution (20 mM KH2PO4; 25:75, v/v) and was delivered at flow rate of 1 mL min−1. The mobile phase was filtered through a nylon membrane filter (0.45 μm pore size, Sigma-Aldrich, Buchs SG, Switzerland) and degassed in an ultrasonic bath (Ultrasonic Cleaner 4200 S2, Soltec S.r.l, Milano, Italy) for 15 min. The injection volume was 20 μL. The detector was set at 248 nm for excitation and 350 nm for emission wavelength. Meanwhile, all the standard and sample solutions were filtered through a cellulose acetate membrane syringe filter (0.2 μm pore size, Advantec MFS Inc, CA, USA) before injection to the chromatographic system. Preparation of standard and sample solutions A stock solution of TRZ (1000 μg mL−1) was prepared monthly by dissolving 100 mg TRZ pure powder in 100 mL deionized water by sonicating for about 2 min. It was stored in a fridge (4°C) until use. Intermediate standard solution (100 μg mL−1) was prepared weekly by dilution of the stock solution. The daily working standard solutions were prepared by serial dilution of intermediate standard solution with phosphate buffer (pH 5.0, 10 mM) to obtain eight working solutions at the concentrations of 0.3–50.0 ng mL−1. All the solutions were filtered through a 0.2 μm cellulose acetate membrane syringe filter before use. Blank human plasma sample was obtained from the Iranian blood transfusion service (Tehran, Iran) and stored at −20°C until use after gentle thawing. Validation samples were prepared daily by spiking 20 μL of working standard solution with different concentrations into 1 mL untreated plasma to obtain final concentrations of 0.3, 1.0, 5.0, 10.0, 20.0, 30.0, 40.0 and 50.0 ng mL−1. Appropriate aliquots of these solutions were transferred into 2 mL conical Eppendorf tubes including 200 μL perchloric acid (15%) as de-proteinating reagent, and the tubes were vortex-mixed for 30 sec at 2000 rpm. The obtained mixtures were centrifuged for 5 min at 12 000 rpm in order to remove excess of proteins. The upper aqueous phase was transferred into new small glass test tubes and diluted with 60 μL of 2 M NaOH. Finally, the filtered supernatants were subjected to the extraction process as optimized. Synthesis of superparamagnetic GO–Fe3O4 nanocomposites GO was synthesized via a modified Hummers method where the amount of KMnO4 (intercalating agent and oxidizing agent) was increased in order to reduce reaction time, and the amount of H2O2 was increased in order to eliminate the excess KMnO4. In this method, the dialysis tubing as an efficient membrane was also used to remove impurities, soluble metal ions and acid residues (for details see Supplementary Data). Fe3O4 MNPs were synthesized by the modified Massart method with some modifications such as increase of reaction temperature to 85°C and pH to 11 in order to the reduce hydroxide ion Fe(OH)2 and Fe(OH)3 (for details see Supplementary Data). The GO–Fe3O4 nanocomposites were prepared according to previously reported impregnation method (28, 29) with some modifications. Briefly, the prepared Fe3O4 MNPs were protonated via dispersion in 1 mol L−1 HNO3 to obtain a positive surface charge and then separated from the reaction medium by applying an external magnetic field. The graphite oxide (0.2 g) was completely dispersed in deionized water (100 mL) and exfoliated to GO by ultrasonication for 2 h. The modified Fe3O4 MNPs (0.4 g) were added to the exfoliated GO solution under a nitrogen atmosphere. After stirring at room temperature for 4 h, the reaction mixture was ultrasonicated for 10 min to form a uniform dispersion. The GO–Fe3O4 nanocomposites were collected by centrifuging and using an external magnetic field to remove the unbounded compounds. Finally, the obtained product was dried in a vacuum oven overnight at 60°C. M-dSPE procedure The M-dSPE procedure was performed as follows. A certain amount of 2 mg GO–Fe3O4 was added into a test tube containing 1 mL plasma sample solution under ultrasonication for 1 min and the pH value was adjusted at 5.0. The mixture solution was shaken on a slow-moving platform shaker for 3 min at ambient temperature in order to reach the adsorption equilibrium. Subsequently, the GO–Fe3O4 was isolated from the sample solution using an external strong magnet and the upper phase was totally discarded. The collected magnetic adsorbent was washed one time with 2 mL deionized water to remove the interferences. Afterwards, 100 μL of 50 mM phosphate buffer pH 10.0 was added as elution solvent, the mixture transferred into a test tube and vigorously shaken for 5 min in order to achieve the maximum recovery of TRZ. The eluate was isolated from the suspension by a strong magnet, and the supernatant was filtered and transferred into a vial. Finally, 20 μL of the sample solution was injected into the HPLC–FL system for analysis. Six replicates for each concentration level were performed. This procedure is shown schematically in Figure 2. Figure 2 Open in new tabDownload slide Schematic illustration of the magnetic-dispersive solid phase extraction procedure using the GO–Fe3O4 sorbent. Figure 2 Open in new tabDownload slide Schematic illustration of the magnetic-dispersive solid phase extraction procedure using the GO–Fe3O4 sorbent. Validation parameters and protocol Validation is a process with the purpose of confirming the qualification and reliability of an analytical method for a specific test. The GO–Fe3O4-based M-dSPE–HPLC–FL method validation was made in terms of typical validation characteristics, such as linearity, selectivity, precision (repeatability), limit of detection (LOD), limit of quantification (LOQ) and accuracy. The linearity of the method was checked by preparing and analyzing eight serial dilutions (0.3, 1.0, 5.0, 10.0, 20.0, 30.0, 40.0 and 50.0 ng mL−1) of the human plasma samples in six replicates (n = 6) for each concentration which were assessed by using calibration curve (slope and intercept) to calculate the coefficient of determination (R2). TRZ calibration curve was created (using Excel) by plotting the average HPLC peak area (Y-axis) ratio versus calibrator concentration (X-axis). The selectivity of the method was assured by analyzing analyte-free human plasma samples using the proposed extraction method and chromatographic conditions in order to rule out any possible interference from endogenous matrix compounds with analyte. Intra- and inter-day precision (expressed as the relative standard deviation, RSD %) and accuracy (expressed as the percentage of the extracted amount, recovery %) of the developed method were assessed by analyzing six replicates (n = 6) spiked plasma samples at three concentration levels (0.3, 20.0 and 50.0 ng mL−1) in a single day as well as between-days (three consecutive days). Extraction recovery was evaluated by comparing the mean peak areas from six replicates matrix-based samples with peak areas of eluent solvent-based samples with equal TRZ concentrations, and the following equation was used to calculate the recovery: [% Recovery = (Found value-Background/Spiked value) × 100]. The LOD was defined as the lowest concentration of TRZ in human plasma samples, which could be readily detected at signal to noise ratio 3:1 (S/N = 3). The LOQ was defined as the lowest plasma concentration of TRZ which could be quantified with a coefficient of variation of less than 20% (30). Results Optimization of the M-dSPE conditions In this study, several parameters affecting M-dSPE performance such as sample pH, the amount of sorbent, extraction time, elution solvent and its volume and desorption time were carefully studied and optimized to achieve the maximal extraction efficiency of GO–Fe3O4 for target analyte (TRZ). Besides, the reusability of the prepared magnetic sorbent in several successive sorption–desorption cycles was investigated. All the optimization studies were carried out for a sample solution containing 20.0 ng mL−1 TRZ, and each experiment was done in three replicates (n = 3) at ambient temperature and average values were used for the following optimization process. Effect of sample pH The sample pH value is one of the most important parameters affecting the extraction performance in the M-dSPE procedure because it determines the molecular or ionic form of the target analytes in aqueous solution and the surface charge density of the sorbent; therefore, this parameter has a key role in the interaction between TRZ and GO–Fe3O4 nanocomposites. In the present work, to investigate the effect of the sample pH on the extraction efficiency (E%) of TRZ (20.0 ng mL−1) by GO–Fe3O4, the pH of sample solution was adjusted to 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0 and 10.0 using appropriate volumes of phosphate buffer solution, while the other experimental conditions were constant (sorbent amount, 2 mg and contact time, 3 min). The results are shown in Figure 3. It was observed that the E% of TRZ significantly increased when the sample pH value was increased from 2 to 5, and then gradually decreased when the pH value was higher than 5.0. The pKa value for TRZ is 7.1. In strong acidic medium (lower pH values), the stability of GO–Fe3O4 decreased and extraction was low (may be due to the electrostatic repulsion between Fe3O4 MNPs and GO nanosheets); in addition, iron oxide was dissolved (31). In strongly alkaline medium (higher pH values), TRZ existed in deprotonated (molecular) form, while the GO–Fe3O4 nanosheets are still negatively charged and thus the interaction between the GO–Fe3O4 surface and analyte was very weak which led to lower extraction of TRZ. At pH 5.0, the stability of the GO–Fe3O4 is high and the attraction between the negative charges of GO functional groups (such as carboxyl and hydroxyl groups) and protonated TRZ is optimal. Therefore, buffer solution of pH 5.0 was chosen as the working pH for the following experiments. Figure 3 Open in new tabDownload slide Effect of sample pH on the extraction efficiency of TRZ (n = 3 for each point). Conditions: TRZ concentration, 20.0 ng mL−1; sample volume, 1 mL; extraction time, 3 min; magnetic sorbent amount, 2 mg; sample pH, 2.0–10.0. Figure 3 Open in new tabDownload slide Effect of sample pH on the extraction efficiency of TRZ (n = 3 for each point). Conditions: TRZ concentration, 20.0 ng mL−1; sample volume, 1 mL; extraction time, 3 min; magnetic sorbent amount, 2 mg; sample pH, 2.0–10.0. Effect of extraction time The M-dSPE technique is an equilibrium-based process; thus, time plays a key role in the extraction of analytes. In the present work, different contact times (1, 2, 3, 4 and 5 min) between sample solution and adsorbent were tested to establish the equilibrium time for maximum extraction while the sample pH was kept 5.0. The results indicated that the extracted amount of TRZ enhanced when the extraction time increased from 1 to 3 min, and then remained almost constant when the extraction time exceeded 3 min. Consequently, the extraction time of 3 min was used for the following studies. The equilibrium adsorption quantity (Qe in mg g−1) of the analyte by the superparamagnetic GO–Fe3O4 nanocomposites was calculated by the following equation: $$ {Q}_e=\left(\left({\mathrm{C}}_0-{\mathrm{C}}_{\mathrm{e}}\right)\mathrm{V}/m\right), $$ where Qe is the amount of TRZ adsorbed per gram of the GO–Fe3O4 at equilibrium (mg g−1), C0 and Ce are initial and equilibrium concentrations of TRZ, respectively, V (L) is the volume of the TRZ solution and m (g) is the weight of the superparamagnetic GO–Fe3O4. In order to determine the adsorption capacity, 10 mg GO–Fe3O4 was added into 5 mL TRZ solutions at concentrations of 1.0–500 mg L−1, and the suspensions were shaken at room temperature for 3 min. Then, the adsorbent were magnetically separated, and the remaining TRZ in the supernatant was measured by HPLC–FL. The adsorption capacity of the GO–Fe3O4 nanocomposite for TRZ was obtained to be about 32.58 mg g−1, which is sufficient to extract trace amounts of TRZ from a large volume sample. Effect of the nano-sorbent amount Compared to ordinary sorbents (micro-sized particles), nano-sized sorbents have larger specific surface area and also exhibit a significantly high extraction capacity and efficiency. Therefore, satisfactory results can be achieved by using fewer amounts of these adsorbents. In order to achieve a high extraction efficiency and recovery, different amounts of the GO–Fe3O4 as adsorbent were tested from 0.5 to 4 mg, while the sample pH was 5.0 and extraction time was set to 3 min. The extraction efficiency of TRZ increased with the increasing sorbent amount up to 2 mg, and then remained almost invariant with further increase. It could be attributed to the enhancement in the surface area and active sites. Although, using large quantities of sorbent is useful for extraction, their removal from solvent extraction are inconvenient in desorption processes. Therefore, in the following studies, 2 mg GO–Fe3O4 was used as the optimum amount of sorbent. Effect of elution solvent type and its volume In the M-dSPE process, to achieve high recovery efficiency as well as to make the extraction process more economical, the selection of an appropriate elution solvent for eluting the analytes from the sorbent is a crucial and considerable step. A good eluting solvent should effectively elute the target compounds from the sorbent with the lowest volume without damaging the nature of the adsorbent surface. In order to select the best desorption eluent for eluting TRZ from the GO–Fe3O4 nanocomposites, different eluent types (acetonitrile (MeCN), ethanol (EtOH), methanol (MeOH), chloroform (Chl), acetone (Ace), 1-propanol (1-PrOH), phosphate buffer pH 2.0 and 10.0) were used and their recovery efficiencies were investigated under the same extraction conditions: TRZ concentration, 20.0 ng mL−1; sample volume, 1 mL; pH value, 5.0; magnetic sorbent amount, 2 mg and extraction time, 3 min. Among the selected solvents, Ace showed low recovery, while 50 mM phosphate buffer pH 10.0 exhibited the highest recovery efficiency for TRZ. It could be attributed to the breaking of the electrostatic interaction between TRZ and GO–Fe3O4 under alkaline conditions. Subsequently, the effect of elution volume on recovery efficiency was tested and optimized in the range of 10–200 μL. As can be seen in Figure 4, with the increase of eluent volume, the recovery efficiency of TRZ increased till 100 μL and then remained almost constant with further increase in volume; therefore, 100 μL was chosen as the optimum volume to ensure high recovery efficiency and preconcentration factor. According to these considerations, 100 μL of 50 mM phosphate buffer pH 10.0 was used as an optimum eluent to complete preconcentration of TRZ in the subsequent determination. Figure 4 Open in new tabDownload slide Effect of volume of elution solvent on the recoveries (n = 3 for each point). Conditions: TRZ concentration, 20.0 ng mL−1; sample volume, 1 mL; pH value, 5.0; extraction time, 3 min; magnetic sorbent amount, 2 mg; elution solvent, solution phosphate buffer pH 10.0; desorption time, 5 min; elution volume, 10–150 μL. Figure 4 Open in new tabDownload slide Effect of volume of elution solvent on the recoveries (n = 3 for each point). Conditions: TRZ concentration, 20.0 ng mL−1; sample volume, 1 mL; pH value, 5.0; extraction time, 3 min; magnetic sorbent amount, 2 mg; elution solvent, solution phosphate buffer pH 10.0; desorption time, 5 min; elution volume, 10–150 μL. Effect of desorption time In order to ensure the complete desorption of the retained analyte from the adsorbent, various desorption times were investigated at different shaking times (1, 2, 3, 4, 5 and 6 min). As shown in Figure 5, the recovery values of TRZ increased with increase in shaking time and then remained constant. According to these results, 5 min was enough for eluting the extracted TRZ from the surface of the GO–Fe3O4 sorbent; therefore, 5 min was chosen as the optimum desorption time. Figure 5 Open in new tabDownload slide Effect of desorption time on the recoveries (n = 3 for each point). Conditions: TRZ concentration, 20.0 ng mL−1; sample volume, 1 mL; pH value, 5.0; extraction time, 3 min; sorbent amount, 2 mg; elution solvent, solution phosphate buffer pH 10.0; elution volume, 100 μL; desorption time, 1–6 min. Figure 5 Open in new tabDownload slide Effect of desorption time on the recoveries (n = 3 for each point). Conditions: TRZ concentration, 20.0 ng mL−1; sample volume, 1 mL; pH value, 5.0; extraction time, 3 min; sorbent amount, 2 mg; elution solvent, solution phosphate buffer pH 10.0; elution volume, 100 μL; desorption time, 1–6 min. Reusability of GO–Fe3O4 nanocomposites To estimate the chemical stability as well as the possibility of regeneration of the superparamagnetic GO–Fe3O4 sorbent, the reusability of the sorbent was investigated through four successive sorption–desorption steps under the optimized conditions. After each step, the sorbent was isolated form the elution solvent using an external magnet and washed with 50 mM phosphate buffer pH 10.0 (100 μL, three times) and deionized water (3 mL, once), respectively. The washed GO–Fe3O4 was then air-dried at room temperature and used directly for the next analysis step under the same conditions. As illustrated in Figure 6, the GO–Fe3O4 sorbent could be effectively reused at least four times without any considerable decrease in its extraction efficiency and magnetism, because it has a vast surface area. The obtained results suggested that the GO–Fe3O4 sorbent is mechanically stable and reusable for the extraction–determination of TRZ from the biological fluids. Figure 6 Open in new tabDownload slide The reusability of the GO–Fe3O4 nanocomposites after four successive uses. Figure 6 Open in new tabDownload slide The reusability of the GO–Fe3O4 nanocomposites after four successive uses. Figure 7 Open in new tabDownload slide Typical chromatograms (HPLC) of (a) blank human plasma, (b) plasma sample spiked with TRZ at concentration of 20.0 ng mL−1 and (c) clean-up with GO–Fe3O4 under optimal conditions. Conditions: column ACE C18 (250 mm × 4.6 mm I.D., 5 μm) at 25°C, eluent acetonitrile:phosphate buffer solution (20 mM KH2PO4) (25:75, v/v) at flow rate of 1 mL min−1, monitoring wavelength: 248–350 nm. Figure 7 Open in new tabDownload slide Typical chromatograms (HPLC) of (a) blank human plasma, (b) plasma sample spiked with TRZ at concentration of 20.0 ng mL−1 and (c) clean-up with GO–Fe3O4 under optimal conditions. Conditions: column ACE C18 (250 mm × 4.6 mm I.D., 5 μm) at 25°C, eluent acetonitrile:phosphate buffer solution (20 mM KH2PO4) (25:75, v/v) at flow rate of 1 mL min−1, monitoring wavelength: 248–350 nm. Table II Comparison of the Proposed Method with Other Methods for the Extraction and Determination of TRZ Method Sample volume Extraction time (min) Linear range (ng ml−1) Recovery (%) RSD% LOD (ng ml−1) LOQ (ng ml−1) Ref. Ionic-liquid microextraction spectrofluorimetry 1 mL/Plasma - 0.1–115 86.5–96.0 2.4 0.27 - (5) 10 mL/Urine 91.5–105.8 Online-SPE–HPLC–UV 1 mL/Plasma - 5.0–500 89.8–96.2 6.5–14.2 0.5 5 (6) LLE–HPLC–UV 1 mL/Plasma 5 10.0–400 99.9–105.2 2.3–6.7 - 10 (7) LLE–HPLC–FL 0.2 mL/Plasma 60 - - - - - (8) LLE–HPLC–FL 1 mL/Plasma 30 1.0–80 94.0–107.0 2.3–10.6 1 - (9) LLE–LC–ESIa–MS 1 mL/Plasma 20 0.062–64 - - - 0.062 (10) GO–Fe3O4–M-dSPE–HPLC–FL 1 mL/Plasma 3 0.3–50.0 97.7–106.0 2.2–3.8 0.09 0.3 This work Method Sample volume Extraction time (min) Linear range (ng ml−1) Recovery (%) RSD% LOD (ng ml−1) LOQ (ng ml−1) Ref. Ionic-liquid microextraction spectrofluorimetry 1 mL/Plasma - 0.1–115 86.5–96.0 2.4 0.27 - (5) 10 mL/Urine 91.5–105.8 Online-SPE–HPLC–UV 1 mL/Plasma - 5.0–500 89.8–96.2 6.5–14.2 0.5 5 (6) LLE–HPLC–UV 1 mL/Plasma 5 10.0–400 99.9–105.2 2.3–6.7 - 10 (7) LLE–HPLC–FL 0.2 mL/Plasma 60 - - - - - (8) LLE–HPLC–FL 1 mL/Plasma 30 1.0–80 94.0–107.0 2.3–10.6 1 - (9) LLE–LC–ESIa–MS 1 mL/Plasma 20 0.062–64 - - - 0.062 (10) GO–Fe3O4–M-dSPE–HPLC–FL 1 mL/Plasma 3 0.3–50.0 97.7–106.0 2.2–3.8 0.09 0.3 This work aElectrospray-ionization. Open in new tab Table II Comparison of the Proposed Method with Other Methods for the Extraction and Determination of TRZ Method Sample volume Extraction time (min) Linear range (ng ml−1) Recovery (%) RSD% LOD (ng ml−1) LOQ (ng ml−1) Ref. Ionic-liquid microextraction spectrofluorimetry 1 mL/Plasma - 0.1–115 86.5–96.0 2.4 0.27 - (5) 10 mL/Urine 91.5–105.8 Online-SPE–HPLC–UV 1 mL/Plasma - 5.0–500 89.8–96.2 6.5–14.2 0.5 5 (6) LLE–HPLC–UV 1 mL/Plasma 5 10.0–400 99.9–105.2 2.3–6.7 - 10 (7) LLE–HPLC–FL 0.2 mL/Plasma 60 - - - - - (8) LLE–HPLC–FL 1 mL/Plasma 30 1.0–80 94.0–107.0 2.3–10.6 1 - (9) LLE–LC–ESIa–MS 1 mL/Plasma 20 0.062–64 - - - 0.062 (10) GO–Fe3O4–M-dSPE–HPLC–FL 1 mL/Plasma 3 0.3–50.0 97.7–106.0 2.2–3.8 0.09 0.3 This work Method Sample volume Extraction time (min) Linear range (ng ml−1) Recovery (%) RSD% LOD (ng ml−1) LOQ (ng ml−1) Ref. Ionic-liquid microextraction spectrofluorimetry 1 mL/Plasma - 0.1–115 86.5–96.0 2.4 0.27 - (5) 10 mL/Urine 91.5–105.8 Online-SPE–HPLC–UV 1 mL/Plasma - 5.0–500 89.8–96.2 6.5–14.2 0.5 5 (6) LLE–HPLC–UV 1 mL/Plasma 5 10.0–400 99.9–105.2 2.3–6.7 - 10 (7) LLE–HPLC–FL 0.2 mL/Plasma 60 - - - - - (8) LLE–HPLC–FL 1 mL/Plasma 30 1.0–80 94.0–107.0 2.3–10.6 1 - (9) LLE–LC–ESIa–MS 1 mL/Plasma 20 0.062–64 - - - 0.062 (10) GO–Fe3O4–M-dSPE–HPLC–FL 1 mL/Plasma 3 0.3–50.0 97.7–106.0 2.2–3.8 0.09 0.3 This work aElectrospray-ionization. Open in new tab Discussion Characterization of GO–Fe3O4 nanocomposites The functional groupings/surface bonding, elemental contents, crystal structure/phase composition, typical morphology and magnetic properties of the synthesized nanomaterials (GO, Fe3O4 MNPs and GO–Fe3O4) were characterized by FT-IR, EDX, XRD, SEM and VSM, respectively (Figures S1–S5, Supplementary Data). The surface electrical potential was quantified through a zeta potential analyzer. In summary, as shown in FT-IR spectra (Figure S1c, Supplementary Data), the GO–Fe3O4 nanocomposites showed an absorption peak at 568 cm−1 corresponding to the Fe–O stretching vibration (for the magnetite phase), which demonstrated that the Fe3O4 MNPs were well loaded on the surface of GO nanosheets. The EDX spectrum analysis (Figure S2c, Supplementary Data) revealed the presence of intense peaks related to iron (72.78%), carbon (20.68%) and oxygen (6.55%) atoms. These data confirmed the purity of the synthesis results. As displayed in Figure S3c, Supplementary Data, the XRD pattern diffraction peaks of GO–Fe3O4 nanocomposites were observed at 2θ = 12.89°, 30.28°, 35.65°, 43.31°, 53.70°, 57.25°, 62.84° and 74.28°, which were ascribed to the (001), (220), (311), (400), (422), (511), (440) and (533) reflections of GO and Fe3O4, respectively. Therefore, the XRD results confirm the successful synthesis of GO–Fe3O4 and no other significant peaks (such as maghemite (γ-Fe2O3) and/or Fe(OH)3 were observed. The SEM image (Figure S4c, Supplementary Data) indicated that the spherical Fe3O4 MNPs with an average particle size of ~32 nm were well distributed on the surface of GO nanosheets (thickness of ~16 nm) by maintaining their particle size distribution and morphology to form the GO–Fe3O4 nanocomposite. According to the VSM results (Figure S5, Supplementary Data), the GO–Fe3O4 nanocomposites showed superparamagnetic behavior at room temperature due to no hysteresis, and the saturation magnetization value (Ms = 56.41 emu g−1) was sufficient for convenient and fast magnetic separation of the adsorbent from the sample solution by an external magnet (as shown in the inset of Figure S5). The surface electric charges of GO and Fe3O4 MNPs were quantified by a nanoparticle zeta potential analyzer at room temperature, and the zeta potentials values were found to be −38.67 and 34.21 mV for GO nanosheets (highly negatively charged due to the presence of various oxygen-containing functional groups) and acidulated Fe3O4 MNPs (in 1 mol L−1 HNO3 to obtain highly positive surface charge), respectively. According to zeta potential measurements, the GO–Fe3O4 nanocomposites were successfully synthesized via a facile one-step electrostatic self-assembly approach. Method validation study Under the optimal experimental conditions, the proposed GO–Fe3O4-based M-dSPE–HPLC–FL method was validated by different quantitative parameters. The linearity range for TRZ determination in human plasma was 0.3–50.0 ng mL−1. The calibration equation of Y = 3984.1X + 1193.4 was obtained with a proper coefficient of determination (R2 = 0.9989), indicating that the relationship between the peak area and TRZ concentration was linear in the considered concentration range. The LOD and LOQ values were found to be 0.09 and 0.3 ng mL−1, respectively. The recovery range of TRZ spiked into human plasma was 97.7–106.6%, and the intra- and inter-day precision (RSD %) were found to be in the range of 2.2–3.8% and 4.7–6.4%, respectively. In addition, the preconcentration factor of 10 was achieved based on the ratio of the slopes of the linear section of the calibration curve before and after preconcentration. Results of validation parameters are listed in Table I. TRZ assay in human plasma samples In order to evaluate the potential of GO–Fe3O4 nanocomposites for the effective clean-up and also applicability of the proposed extraction procedure for complicated samples, the validated GO–Fe3O4-based M-dSPE–HPLC–FL method was established and applied to the preconcentration and determination of TRZ in the human plasma samples. The typical chromatograms of (i) drug free human plasma sample, (ii) plasma sample spiked with 20.0 ng mL−1 of TRZ without cleaned up by M-dSPE and (iii) plasma sample spiked with 20.0 ng mL−1 of TRZ with cleaned up by M-dSPE under optimized conditions are shown in Figure 7. Before extraction, the endogenous interfering peaks were observed at about 2.55 and 3.33 min in the chromatogram of plasma samples. The retention time of TRZ was 3.85 min with the total run-time of 7 min. Moreover, the Empower software was applied to determine TRZ peak purity (selectivity) by comparing the purity threshold and purity angle which demonstrated that the method was specific for TRZ. Comparison of GO–Fe3O4-based M-dSPE method with other methods The performance of the proposed method for the analysis of TRZ in human biological samples in terms of extraction time, linear range, LOD, LOQ, RSD and recovery were compared with previous reported methods. The comparison results are listed in Table II. In the majority of these reported methods, LLE of TRZ from biological samples was performed with consuming large volume of toxic and expensive organic solvents followed by tedious chemical procedures and a time-consuming evaporation step. An online SPE method-based weak cation-exchange (WCX) column (6) demands much less quantities of organic solvents than LLE, but it is costly, labor intensive and includes several steps, also, it shows lower sensitivity than the present method. A radioreceptor assay method described by Taguchi et al. (8) is too complex to be applied in routine pharmacokinetic studies and may not be selective to TRZ due to the possibility of receptor binding by its metabolites. The reported LC–ESI–MS method (10) showed good sensitivity, but such a detector system may not be available in most laboratories and also not suitable for routine clinical analysis. In comparison with the listed methods (Table II), the proposed method had shorter extraction time and the separation procedure was fast and convenient without the need for centrifugation or filtration step, and also a small amount of non-toxic extraction solvents were consumed. It is clearly seen from Table II that the proposed method exhibited an applicable linear range and LOQ, adequate sensitivity, good accuracy and precision. Conclusion In this study, the superparamagnetic GO–Fe3O4 with good stability and reusability was successfully synthesized, characterized and then used as a potential adsorbent in a simple and robust M-dSPE clean-up technique for the determination of trace amounts of TRZ in human plasma samples prior to HPLC–FL analysis. Since nanoparticle sorbents possess very high surface areas and a short diffusion route, high extraction efficiency can be achieved in a very short extraction time. This developed method provided effectively clean extracts and omitted interfering peaks from the human plasma matrix. It was rapid, cost-effective, convenient, environment-friendly, with a very low consumption of sorbent (2 mg) and elution solvent (100 μL). Besides, the good repeatability, precision, preconcentration factor, satisfactory linear response and acceptable recoveries were obtained. 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TI - Magnetic-Dispersive Solid Phase Extraction Based on Graphene Oxide–Fe3O4 Nanocomposites Followed by High Performance Liquid Chromatography-Fluorescence for the Preconcentration and Determination of Terazosin Hydrochloride in Human Plasma
JF - Journal of Chromatographic Science
DO - 10.1093/chromsci/bmz085
DA - 2020-01-23
UR - https://www.deepdyve.com/lp/oxford-university-press/magnetic-dispersive-solid-phase-extraction-based-on-graphene-oxide-1hp51dKSTP
SP - 1
VL - Advance Article
IS - 
DP - DeepDyve
ER -