Research article Received: 11 December 2014,
Revised: 27 April 2015,
Accepted: 7 May 2015
Published online in Wiley Online Library
(wileyonlinelibrary.com) DOI 10.1002/bmc.3509
Magnetic solid-phase extraction for determination of sulpiride in human urine and blood using high-performance liquid chromatography Jiao Zhaoa,b, Wenlong Liaoa and Yaling Yanga* ABSTRACT: A novel and efficient sample preconcentration technique based on the Fe3O4 magnetic nanoparticles (Fe3O4 MNPs) coated with silica (SiO2) has been developed for extraction and determination of sulpiride. The functionalized MNPs showed excellent dispersibility in aqueous solution and were applied to magnetic solid-phase extraction of sulpiride from human urine and blood prior to high-performance liquid chromatography analysis. The separation, preconcentration and desorption procedure was completed in 10 min. Optimal experimental conditions, including sample pH, the amount of the MNPs, eluent type and volume, and the ultrasonication time were studied and established. The method showed good linearity for the determination of sulpiride in the concentration range of 10–1000 ng/mL in urine and blood. The recovery of the method was in the range between 91.2 and 97.5%, and the limit of detection was 2 ng/mL for sulpiride in human blood and urine. The results indicated that the present procedure is a suitable pretreatment method for biological samples. Copyright © 2015 John Wiley & Sons, Ltd. Keywords: sulpiride; Fe3O4 magnetic nanoparticles; urine; blood; high performance liquid chromatography
Introduction Sulpiride is an antipsychotic drug belonging to the substituted benzamide group. It has been widely used in humans because it has antipsychotic, antidepressive and antiulcer effects with a low frequency of extrapyramidal side-effects (Meyer-Lindenberg et al., 1997). The daily dose for these indications is 200–800 mg (Rzewuska, 1998). It is also prescribed at doses of 50–150 mg in human for the treatment of gastric or duodenal ulcers (Desai and Parmar, 1994), in the treatment of an irritable colon owing to psychosomatic stress (Lanfranchi et al., 1983), and in various vertigo syndromes (Zanetti et al., 2004). Sulpiride can be slowly and poorly absorbed from the gastrointestinal tract with peak serum levels occurring in 2–6 h (Alam et al., 1980); they are also partly excreted by kidney. Several analytical methods have been developed recently for determination of sulpiride, including spectrophotometry (Bressolle et al., 1979; el Walily et al., 1999), spectrofluorometric determination (Bano et al., 2011), mass spectrometric detection (Danielle et al., 2013), adsorptive stripping voltammetry (Farghaly, 2000), high-performance liquid chromatography with ultraviolet detection (Naguib and Abdelkawy, 2010) and with fluorescence detection (Walash et al., 2012). However, the concentration of sulpiride is too low to be quantified in biological samples directly. Thus, sample pretreatment of biological samples has become critical prior to chemical analysis, and several new methods have been developed during the past 10 years, including pressurised liquid extraction (Runnqvist et al., 2010), solid-phase extraction (Gasperotti et al., 2014) and solid-phase micro-extraction (Yang and Xie, 2006). However, these methods are little used for analysis of biological samples because of the complicated matrix and small quantity of sample. Many sample cleanup procedures are usually required to remove matrix
Biomed. Chromatogr. 2015
components that may interfere with the analysis (Fuoco et al., 2005; Kiplagat et al., 2011; Fernández-Ramos et al., 2014). Magnetic nanoparticles (MNPs) such as magnetite (Fe3O4) (Shen et al., 2014) and maghemite (γ-Fe2O3) (Lin et al., 2012) possess high surface areas, which can improve the adsorption capacity of the analyte and superparamagnetism properties, allowing convenient separation of adsorbent from the solution by applying a strong magnetic field in a batch system. These properties make the MNPs an excellent candidate as solid-phase extractor for extraction and separation of various analytes. However, free MNPs such as nano-magnetic iron oxide particles are characterized by some limitations such as hydrophobic surface properties that limit their dispersion into aqueous solutions, and particles that aggregate easily under the action of gravity and strong magnetic attractions (Mahmoud et al., 2013). The nontoxic silica is an ideal coating material because of its capability to form extensive cross-linking, which leads to an inert outer shield that can overcome the limitations encountered by these magnetic nanosorbents. Further, silanized nanocomposites are stable in a wide range of biological environments, including physiological * Correspondence to: Y. Yang, Faculty of Life Science and Technology, Kunming University of Science and Technology, Kunming 650500, China. Email: [email protected] a
Faculty of Life Science and Technology, Kunming University of Science and Technology, Kunming, 650500, China
b
Faculty of Environmental Science and Engineering, Kunming University of Science and Technology, Kunming, 650500, China Abbreviations used: MNP, magnetic nanoparticle; MSPE, magnetic solidphase extraction.
Copyright © 2015 John Wiley & Sons, Ltd.
J. Zhao et al. and supraphysiological salt concentrations. They are biocompatible and can also be easily activated to provide new functional groups (Du et al., 2006). Functionalized MNPs make performance of the separation process directly in biological samples containing haemoglobin, proteins and fatty posible without the need for additional deproteinization, deesterification and decoloration (de Loor et al., 2008; Liu et al., 2010), which makes separation easier and faster. In the present paper, surface modification of Fe3O4 maghemite nanoparticles with silica particles can afford additional applications via attached surface Si–OH bonding groups (Mahmoud et al., 2013). For the presence of imido of sulpiride, the chemical interaction between Fe3O4@SiO2 and sulpiride may be due to the formation of intramolecular hydrogen bonding. In addition, the highest determined sulpiride sorption capacity value was found at pH 7.0; adsorption of sulpiride to Fe3O4–SiO2 was probably based on covalent bonding ‘chemisorption’ produced by the nitrogen donor atoms located within the structure of sulpiride and surface oxygen donor functional groups of the sorbent. The magnetic solid-phase extraction (MSPE) technique was found to be very useful and cost-effective for a better preconcentration and determination of the sulpiride in the urine and blood treatment.
Quantification of sulpiride in urine and blood samples Apparatus Chromatographic separation and evaluation were performed on an HPLC system consisting of a vacuum degasser, an autosampler, a quaternary pump, and a diode-array detector (Agilent 1200 Series, Agilent Technologies, California, USA). A PHS-3 (Shanghai, China) pH meter was used for pH measurements. A water bath (Shanghai, China) was used to maintain solutions at certain temperatures. A vacuum drying oven BPZ-6033 (Shanghai, China) was used to dry synthesized nanomaterials. An ultrasonic cleaner (Shanghai, China) was used for ultrasound-assisted extraction. HPLC conditions The separation was performed on an Agilent TC-C18 column (150 × 4.6 mm, i.d., 5 μm). An Agilent Chemstation for LC system was utilized to control the system and for the acquisition and analysis of the chromatographic data. Quantification was done by the evaluation of peak areas. The column temperature was maintained at 30 °C. Acetonitrile–methanol–water solution containing 6.8 g/L of potassium dihydrogen phosphate and 1 g/L of sodium octanesulfonate adjusted to pH 3.3 using phosphoric acid (10:10:80%, v/v/v) were used for the isocratic elution of the sulpiride. The flow rate of mobile phase was 1.0 mL/min. The injection volume was 10 μL, and the Diode Array Detector (DAD) detector was chosen at 240 nm. Chemical reagents All reagents were at least at analytical grade unless otherwise noted. Sulpiride (Figure 1) (European Pharmacopoeia Reference Standard) was purchased from Strasbourg Cedex. Standard stock solutions of sulpiride were prepared in methanol at a concentration of 500 mg/mL. Working solutions were prepared daily by an
wileyonlinelibrary.com/journal/bmc
Figure 1. Structure of sulpiride.
appropriate dilution of the stock solutions. Acetonitrile and methanol were chromatographic (Tedia Company, USA). Preparation of blood samples The whole blood samples were obtained from 10 volunteers who were on sulpiride treatment for 1 month in the First People’s Hospital of Yunnan Province (Kunming, China). The 5 mL blood samples were collected in disposable vacuum blood specimen collection tubes containing anticoagulant. Plasma in the upper layer was transferred to a new tube after each whole blood sample was centrifuged at 2000 rpm for 10 min. The plasma was diluted to a volume of 5 mL with distilled water for further analysis according to extraction procedure. Preparation of urine samples The urine samples were collected from 10 adult volunteers who had been on sulpiride treatment for 1 month in the First People’s Hospital of Yunnan Province (Kunming, China). The urine samples were collected in disposable polypropylene specimen cups. A 5 mL aliquot of urine was transferred to a new tube for further analysis according to the extraction procedure. Synthesis of silica-coated magnetite nanoparticles Fe3O4/SiO2 The Fe3O4 MNPs were synthesized using the co-precipitation method (Wang et al., 2010; Lu et al., 2007). A 5.0 g aliquot of FeCl2 · 4H2O and 13.6 g of FeCl3 · 6H2O were dissolved in 50 mL of deionized water. The mixture was added dropwise into 50 mL buffer solution (pH 10) of ammonia solution/ammonium nitrate in a waterbath at 80 °C and 10 mL ammonia solution was added into the reaction solution under vigorous stirring and nitrogen gas protection for 30 min. The formed Fe3O4 nanoparticles were separated from the medium by the action of an external magnetic field, and washed for four times with distilled water followed by drying at 40 °C for 24 h. The Fe3O4 NPs were functionalized with SiO2 according to the process as follows: 1.0 g of dried Fe3O4 NPs was suspended in 100.0 mL distilled water at 80 °C. 10.0 mL of 1.0 mol/L sodium silicate solution was added dropwise to this suspension under vigorous stirring for 3.0 h under nitrogen gas protection and the pH of this mixture was adjusted to 6.0. The obtained Fe3O4@ SiO2 naniparticles were collected with an external supermagnet and washed with distilled water for four times, and then diluted to 20 mL with deionized water and stored in a freezer (4 °C) for further use. The concentration of Fe3O4@ SiO2 suspension was about 50 mg/mL. MSPE procedure The MSPE procedure was carried out as follows: first, 80 μL of Fe3O4@SiO2 MNPs was added to a 5 mL sample solution. Second, ultrasound was applied to diffuse the MNPs in the sample solution
Copyright © 2015 John Wiley & Sons, Ltd.
Biomed. Chromatogr. 2015
MSPE for determination of sulpiride in human urine and blood by HPLC 100
80
Recovery(%)
for 4 min. After MNPs had been isolated from the solution with a strong magnet at the bottom of the tube and the supernatant had been poured away, 0.02 M NaOH was used as eluent for desorption of adsorbed sulpiride. The particles were separated again by positioning a magnet to the outside of tube wall so that the eluate solution could be completely removed with a syringe and then filtered through HNWP nylon membranes (0.45 μm, Millipore, Bedford, MA, USA) prior to HPLC analysis. A 20.0 μL aliquot of isolated eluate was injected into the HPLC system for analysis. Figure 2 showed the phenomena of Fe3O4@ SiO2 dispersed in plasma sample solution (a) and collected by external magnet (b). The procedures of adsorption and separation could be finished within 10 min.
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Figure 3. Effect of pH. Extraction conditions: sample volume, 5.0 mL spiked with 200 ng/mL of sulpiride; magnetic nanoparticles (MNPs), 4 mg; ultrasonication time, 4 min; eluent solvent, 0.2 mL of 0.02 mol/L NaOH.
Results and discussion Optimization of the MSPE procedure In order to obtain the best analytical performance, 5.0 mL of prepared human urine and plasma spiked with 200 ng/mL of sulpiride were used. The influence of different experimental parameters, including sample pH, eluent type and volume, the amount of the MNPs and the ultrasonication time, on the performance of the extraction procedure weas determined. The recovery of sulpiride was used to evaluate the extraction efficiency under different experimental conditions. All the experiments were performed five times and the averages of the results were used for optimization. Blank urine and plasma were obtained from a healthy volunteer who was not taking sulpiride and also run under the same procedure. Effect of solution pH Solution pH plays an important role for the adsorption of the analytes and the analytes should be electrically neutral so that they can be efficiently adsorbed and the adsorption is unaffected by charges on the surface of the sorbent. To evaluate the effect of sample pH on the recovery of the sulpiride, the pH of the solution varied in the range of 4.0–11.0 using 0.1 mol/L HCl or NaOH solutions for pH adjustment. As shown in Fig. 3, the recovery increased with increasing pH and reached maximum at pH 6.0–8.0,
decreasing at higher pH values. Since the pH of the urine and plasma samples was generally around 7, it was unnecessary to adjust the pH of the sample solution. Effect of the amount of MNPs To investigate the effect of MNPs amount on the extraction efficiency, different amounts of adsorbent amount over the range of 1–5 mg were added to the solution. Based on the results as shown in Fig. 4, the recovery of sulpiride increased with increase in MNPs amount up to 4 mg, and then it remained constant. Fe3O4 magnetite nanoparticles modified with SiO2 is a novel nanosorbent that combines the magnetic characteristics owing to the existence of MNPs with high surface areas like nano-silicon oxide for extraction of the analyte. Therefore, smaller amounts of adsorbent can achieve satisfactory results and 4 mg of adsorbent was used in all experiments. Effect of ultrasonication time In the process of adsorption, the contact time is one of the prime factors influencing the target analyte extraction. When the MNPs were separated immediately without a contact process into the sample, the recovery of sulpiride was only 35%. Ultrasound can 100
Recovery(%)
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Figure 2. Photographs of Fe3O4@ SiO2 dispersed in sample solution (a) and collected by supermagnet (b).
Biomed. Chromatogr. 2015
Figure 4. Effect of the amount of MNPs. Extraction conditions: sample volume, 5.0 mL spiked with 200 ng/mL of sulpiride; pH 7.0; MNPs, 4 mg; ultrasonication time, 4 min; eluent solvent, 0.2 mL of 0.02 mol/L NaOH.
Copyright © 2015 John Wiley & Sons, Ltd.
wileyonlinelibrary.com/journal/bmc
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Recovery(%)
J. Zhao et al.
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0 Acetonitrile
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Figure 5. Effect of ultrasonication time. Extraction conditions: sample volume, 5.0 mL spiked with 200 ng/mL of sulpiride; pH 7.0; MNPs, 4 mg; eluent solvent, 0.2 mL of 0.02 mol/L NaOH.
accelerate the interactive rate between the MNPs and aqueous phase so that the target analytes could be well adsorbed on the surface of MNPs in a shorter time. The effect of ultrasonication time in the range of 0–10 min was evaluated at 25 Hz of ultrasonication frequency to reveal the effect of extraction time on the recovery of the sulpiride. As indicated in Fig. 5, the extraction recovery of the sulpiride reached a maximum at 4 min. Therefore, 4 min was chosen for the following experiments.
Effect of type and volume of eluent solvent In order to desorb the sulpiride from MNPs surfaces and obtain a high enrichment factor, a suitable eluent should be used and the sorbed analyte should be desorbed in a small volume. Various eluent solvents such as ethanol, acetonitrile, methanol, acetone and sodium hydroxide solution were investigated. As shown in Fig. 6, the highest extraction recovery of sulpiride was achieved using 0.02 mol/L NaOH solutions as the desorbing solvent, which could be explained by the sulpiride having a better solubility in NaOH solution than other solvents. The effect of eluent volume was also tested by eluting sulpiride with the range of 0.1–0.5 mL of NaOH. It was observed that the recovery of sulpiride was increased with increasing the volume NaOH from 0.1 to 0.5 mL. A 0.2 mL aliquot of 0.02 mol/L NaOH was used for the elution of the adsorbed sulpiride in subsequent experiments.
Figure 6. Effect of eluent solvent. Extraction conditions: sample volume, 5.0 mL spiked with 200 ng/mL of sulpiride; pH 7.0; MNPs, 4 mg; ultrasonication time, 4 min; eluent solvent, 0.2 mL of 0.02 mol/L NaOH.
Effect of other molecules The optimal experimental conditions described above were used to study the inhibitory effect of biological amines on the extractability of the proposed method, such as arginine, ascorbic acid, aspartic acid, L-valine, L-leucine and glycine. In addition, some other analog antipsychotic drugs including benzamide, tiapride, chlorpromazine hydrochloride and doxepin were added to the sample solutions. As shown in Table 1, an equimolar concentration of each compound had no significant influence on the adsorption of sulpiride in urine and blood samples, while at a higher molar excess (10:1) of these compounds, a slight decrease of adsorption efficiency was observed. This indicates that the selectivity of the adsorption of sulpiride to MNPs is very high.
Comparison with different MNPs The application of the proposed synthetic Fe3O4@SiO2 MNPs to extract the sulpiride from human urine and blood for HPLC/DAD analysis was compared with Fe3O4 MNPs and conventional HPLC/DAD analysis. Figure 7 shows a comparison of chromatograms of analysis of sulpiride in human urine and blood samples by HPLC/DAD (A) without pre-treatment, and by MSPE-HPLC/ DAD using Fe3O4 (B) or Fe3O4@SiO2 (C) as the extraction solvent. Magnetic nanoparticles have excellent extraction efficiency of sulpiride in biological fluids, and the extraction efficiency of
Table 1. Effect of other molecules on the adsorption of sulpiride from urine and blood samples Analyte
100 ng/mL sulpiride 100 ng/mL sulpiride 100 ng/mL sulpiride
Coexisting compounds
— 100 ng/mL biological aminesa 1000 ng/mL biological amines 100 ng/mL analog drugsb 1000 ng/mL analog drugs
Recovery (%)
RSD (%) (n = 6)
Urine
Blood
Urine
Blood
92.8 89.2 80.5 90.3 82.6
90.9 88.7 82.9 87.8 79.2
3.9 5.9 7.1 5.5 6.9
4.6 5.4 5.9 7.3 7.2
a
Biological components were arginine, ascorbic acid, aspartic acid, glycine, L-valine and L-leucine Analog drugs were benzamide, tiapride, doxepin and chlorpromazine hydrochloride.
b
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Copyright © 2015 John Wiley & Sons, Ltd.
Biomed. Chromatogr. 2015
MSPE for determination of sulpiride in human urine and blood by HPLC mAU
A.HPLC/DAD
sulpiride from urine and blood. The reproducibility of the nanoparticles was described as the recovery of standard sulpiride added to human urine and blood. Then the intra-assay variation of the nanoparticles was measured, the RSD of which was
Revised: 27 April 2015,
Accepted: 7 May 2015
Published online in Wiley Online Library
(wileyonlinelibrary.com) DOI 10.1002/bmc.3509
Magnetic solid-phase extraction for determination of sulpiride in human urine and blood using high-performance liquid chromatography Jiao Zhaoa,b, Wenlong Liaoa and Yaling Yanga* ABSTRACT: A novel and efficient sample preconcentration technique based on the Fe3O4 magnetic nanoparticles (Fe3O4 MNPs) coated with silica (SiO2) has been developed for extraction and determination of sulpiride. The functionalized MNPs showed excellent dispersibility in aqueous solution and were applied to magnetic solid-phase extraction of sulpiride from human urine and blood prior to high-performance liquid chromatography analysis. The separation, preconcentration and desorption procedure was completed in 10 min. Optimal experimental conditions, including sample pH, the amount of the MNPs, eluent type and volume, and the ultrasonication time were studied and established. The method showed good linearity for the determination of sulpiride in the concentration range of 10–1000 ng/mL in urine and blood. The recovery of the method was in the range between 91.2 and 97.5%, and the limit of detection was 2 ng/mL for sulpiride in human blood and urine. The results indicated that the present procedure is a suitable pretreatment method for biological samples. Copyright © 2015 John Wiley & Sons, Ltd. Keywords: sulpiride; Fe3O4 magnetic nanoparticles; urine; blood; high performance liquid chromatography
Introduction Sulpiride is an antipsychotic drug belonging to the substituted benzamide group. It has been widely used in humans because it has antipsychotic, antidepressive and antiulcer effects with a low frequency of extrapyramidal side-effects (Meyer-Lindenberg et al., 1997). The daily dose for these indications is 200–800 mg (Rzewuska, 1998). It is also prescribed at doses of 50–150 mg in human for the treatment of gastric or duodenal ulcers (Desai and Parmar, 1994), in the treatment of an irritable colon owing to psychosomatic stress (Lanfranchi et al., 1983), and in various vertigo syndromes (Zanetti et al., 2004). Sulpiride can be slowly and poorly absorbed from the gastrointestinal tract with peak serum levels occurring in 2–6 h (Alam et al., 1980); they are also partly excreted by kidney. Several analytical methods have been developed recently for determination of sulpiride, including spectrophotometry (Bressolle et al., 1979; el Walily et al., 1999), spectrofluorometric determination (Bano et al., 2011), mass spectrometric detection (Danielle et al., 2013), adsorptive stripping voltammetry (Farghaly, 2000), high-performance liquid chromatography with ultraviolet detection (Naguib and Abdelkawy, 2010) and with fluorescence detection (Walash et al., 2012). However, the concentration of sulpiride is too low to be quantified in biological samples directly. Thus, sample pretreatment of biological samples has become critical prior to chemical analysis, and several new methods have been developed during the past 10 years, including pressurised liquid extraction (Runnqvist et al., 2010), solid-phase extraction (Gasperotti et al., 2014) and solid-phase micro-extraction (Yang and Xie, 2006). However, these methods are little used for analysis of biological samples because of the complicated matrix and small quantity of sample. Many sample cleanup procedures are usually required to remove matrix
Biomed. Chromatogr. 2015
components that may interfere with the analysis (Fuoco et al., 2005; Kiplagat et al., 2011; Fernández-Ramos et al., 2014). Magnetic nanoparticles (MNPs) such as magnetite (Fe3O4) (Shen et al., 2014) and maghemite (γ-Fe2O3) (Lin et al., 2012) possess high surface areas, which can improve the adsorption capacity of the analyte and superparamagnetism properties, allowing convenient separation of adsorbent from the solution by applying a strong magnetic field in a batch system. These properties make the MNPs an excellent candidate as solid-phase extractor for extraction and separation of various analytes. However, free MNPs such as nano-magnetic iron oxide particles are characterized by some limitations such as hydrophobic surface properties that limit their dispersion into aqueous solutions, and particles that aggregate easily under the action of gravity and strong magnetic attractions (Mahmoud et al., 2013). The nontoxic silica is an ideal coating material because of its capability to form extensive cross-linking, which leads to an inert outer shield that can overcome the limitations encountered by these magnetic nanosorbents. Further, silanized nanocomposites are stable in a wide range of biological environments, including physiological * Correspondence to: Y. Yang, Faculty of Life Science and Technology, Kunming University of Science and Technology, Kunming 650500, China. Email: [email protected] a
Faculty of Life Science and Technology, Kunming University of Science and Technology, Kunming, 650500, China
b
Faculty of Environmental Science and Engineering, Kunming University of Science and Technology, Kunming, 650500, China Abbreviations used: MNP, magnetic nanoparticle; MSPE, magnetic solidphase extraction.
Copyright © 2015 John Wiley & Sons, Ltd.
J. Zhao et al. and supraphysiological salt concentrations. They are biocompatible and can also be easily activated to provide new functional groups (Du et al., 2006). Functionalized MNPs make performance of the separation process directly in biological samples containing haemoglobin, proteins and fatty posible without the need for additional deproteinization, deesterification and decoloration (de Loor et al., 2008; Liu et al., 2010), which makes separation easier and faster. In the present paper, surface modification of Fe3O4 maghemite nanoparticles with silica particles can afford additional applications via attached surface Si–OH bonding groups (Mahmoud et al., 2013). For the presence of imido of sulpiride, the chemical interaction between Fe3O4@SiO2 and sulpiride may be due to the formation of intramolecular hydrogen bonding. In addition, the highest determined sulpiride sorption capacity value was found at pH 7.0; adsorption of sulpiride to Fe3O4–SiO2 was probably based on covalent bonding ‘chemisorption’ produced by the nitrogen donor atoms located within the structure of sulpiride and surface oxygen donor functional groups of the sorbent. The magnetic solid-phase extraction (MSPE) technique was found to be very useful and cost-effective for a better preconcentration and determination of the sulpiride in the urine and blood treatment.
Quantification of sulpiride in urine and blood samples Apparatus Chromatographic separation and evaluation were performed on an HPLC system consisting of a vacuum degasser, an autosampler, a quaternary pump, and a diode-array detector (Agilent 1200 Series, Agilent Technologies, California, USA). A PHS-3 (Shanghai, China) pH meter was used for pH measurements. A water bath (Shanghai, China) was used to maintain solutions at certain temperatures. A vacuum drying oven BPZ-6033 (Shanghai, China) was used to dry synthesized nanomaterials. An ultrasonic cleaner (Shanghai, China) was used for ultrasound-assisted extraction. HPLC conditions The separation was performed on an Agilent TC-C18 column (150 × 4.6 mm, i.d., 5 μm). An Agilent Chemstation for LC system was utilized to control the system and for the acquisition and analysis of the chromatographic data. Quantification was done by the evaluation of peak areas. The column temperature was maintained at 30 °C. Acetonitrile–methanol–water solution containing 6.8 g/L of potassium dihydrogen phosphate and 1 g/L of sodium octanesulfonate adjusted to pH 3.3 using phosphoric acid (10:10:80%, v/v/v) were used for the isocratic elution of the sulpiride. The flow rate of mobile phase was 1.0 mL/min. The injection volume was 10 μL, and the Diode Array Detector (DAD) detector was chosen at 240 nm. Chemical reagents All reagents were at least at analytical grade unless otherwise noted. Sulpiride (Figure 1) (European Pharmacopoeia Reference Standard) was purchased from Strasbourg Cedex. Standard stock solutions of sulpiride were prepared in methanol at a concentration of 500 mg/mL. Working solutions were prepared daily by an
wileyonlinelibrary.com/journal/bmc
Figure 1. Structure of sulpiride.
appropriate dilution of the stock solutions. Acetonitrile and methanol were chromatographic (Tedia Company, USA). Preparation of blood samples The whole blood samples were obtained from 10 volunteers who were on sulpiride treatment for 1 month in the First People’s Hospital of Yunnan Province (Kunming, China). The 5 mL blood samples were collected in disposable vacuum blood specimen collection tubes containing anticoagulant. Plasma in the upper layer was transferred to a new tube after each whole blood sample was centrifuged at 2000 rpm for 10 min. The plasma was diluted to a volume of 5 mL with distilled water for further analysis according to extraction procedure. Preparation of urine samples The urine samples were collected from 10 adult volunteers who had been on sulpiride treatment for 1 month in the First People’s Hospital of Yunnan Province (Kunming, China). The urine samples were collected in disposable polypropylene specimen cups. A 5 mL aliquot of urine was transferred to a new tube for further analysis according to the extraction procedure. Synthesis of silica-coated magnetite nanoparticles Fe3O4/SiO2 The Fe3O4 MNPs were synthesized using the co-precipitation method (Wang et al., 2010; Lu et al., 2007). A 5.0 g aliquot of FeCl2 · 4H2O and 13.6 g of FeCl3 · 6H2O were dissolved in 50 mL of deionized water. The mixture was added dropwise into 50 mL buffer solution (pH 10) of ammonia solution/ammonium nitrate in a waterbath at 80 °C and 10 mL ammonia solution was added into the reaction solution under vigorous stirring and nitrogen gas protection for 30 min. The formed Fe3O4 nanoparticles were separated from the medium by the action of an external magnetic field, and washed for four times with distilled water followed by drying at 40 °C for 24 h. The Fe3O4 NPs were functionalized with SiO2 according to the process as follows: 1.0 g of dried Fe3O4 NPs was suspended in 100.0 mL distilled water at 80 °C. 10.0 mL of 1.0 mol/L sodium silicate solution was added dropwise to this suspension under vigorous stirring for 3.0 h under nitrogen gas protection and the pH of this mixture was adjusted to 6.0. The obtained Fe3O4@ SiO2 naniparticles were collected with an external supermagnet and washed with distilled water for four times, and then diluted to 20 mL with deionized water and stored in a freezer (4 °C) for further use. The concentration of Fe3O4@ SiO2 suspension was about 50 mg/mL. MSPE procedure The MSPE procedure was carried out as follows: first, 80 μL of Fe3O4@SiO2 MNPs was added to a 5 mL sample solution. Second, ultrasound was applied to diffuse the MNPs in the sample solution
Copyright © 2015 John Wiley & Sons, Ltd.
Biomed. Chromatogr. 2015
MSPE for determination of sulpiride in human urine and blood by HPLC 100
80
Recovery(%)
for 4 min. After MNPs had been isolated from the solution with a strong magnet at the bottom of the tube and the supernatant had been poured away, 0.02 M NaOH was used as eluent for desorption of adsorbed sulpiride. The particles were separated again by positioning a magnet to the outside of tube wall so that the eluate solution could be completely removed with a syringe and then filtered through HNWP nylon membranes (0.45 μm, Millipore, Bedford, MA, USA) prior to HPLC analysis. A 20.0 μL aliquot of isolated eluate was injected into the HPLC system for analysis. Figure 2 showed the phenomena of Fe3O4@ SiO2 dispersed in plasma sample solution (a) and collected by external magnet (b). The procedures of adsorption and separation could be finished within 10 min.
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Figure 3. Effect of pH. Extraction conditions: sample volume, 5.0 mL spiked with 200 ng/mL of sulpiride; magnetic nanoparticles (MNPs), 4 mg; ultrasonication time, 4 min; eluent solvent, 0.2 mL of 0.02 mol/L NaOH.
Results and discussion Optimization of the MSPE procedure In order to obtain the best analytical performance, 5.0 mL of prepared human urine and plasma spiked with 200 ng/mL of sulpiride were used. The influence of different experimental parameters, including sample pH, eluent type and volume, the amount of the MNPs and the ultrasonication time, on the performance of the extraction procedure weas determined. The recovery of sulpiride was used to evaluate the extraction efficiency under different experimental conditions. All the experiments were performed five times and the averages of the results were used for optimization. Blank urine and plasma were obtained from a healthy volunteer who was not taking sulpiride and also run under the same procedure. Effect of solution pH Solution pH plays an important role for the adsorption of the analytes and the analytes should be electrically neutral so that they can be efficiently adsorbed and the adsorption is unaffected by charges on the surface of the sorbent. To evaluate the effect of sample pH on the recovery of the sulpiride, the pH of the solution varied in the range of 4.0–11.0 using 0.1 mol/L HCl or NaOH solutions for pH adjustment. As shown in Fig. 3, the recovery increased with increasing pH and reached maximum at pH 6.0–8.0,
decreasing at higher pH values. Since the pH of the urine and plasma samples was generally around 7, it was unnecessary to adjust the pH of the sample solution. Effect of the amount of MNPs To investigate the effect of MNPs amount on the extraction efficiency, different amounts of adsorbent amount over the range of 1–5 mg were added to the solution. Based on the results as shown in Fig. 4, the recovery of sulpiride increased with increase in MNPs amount up to 4 mg, and then it remained constant. Fe3O4 magnetite nanoparticles modified with SiO2 is a novel nanosorbent that combines the magnetic characteristics owing to the existence of MNPs with high surface areas like nano-silicon oxide for extraction of the analyte. Therefore, smaller amounts of adsorbent can achieve satisfactory results and 4 mg of adsorbent was used in all experiments. Effect of ultrasonication time In the process of adsorption, the contact time is one of the prime factors influencing the target analyte extraction. When the MNPs were separated immediately without a contact process into the sample, the recovery of sulpiride was only 35%. Ultrasound can 100
Recovery(%)
90
80
70
60
50 1
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4
5
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Figure 2. Photographs of Fe3O4@ SiO2 dispersed in sample solution (a) and collected by supermagnet (b).
Biomed. Chromatogr. 2015
Figure 4. Effect of the amount of MNPs. Extraction conditions: sample volume, 5.0 mL spiked with 200 ng/mL of sulpiride; pH 7.0; MNPs, 4 mg; ultrasonication time, 4 min; eluent solvent, 0.2 mL of 0.02 mol/L NaOH.
Copyright © 2015 John Wiley & Sons, Ltd.
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100
100
80
80
Recovery(%)
Recovery(%)
J. Zhao et al.
60
40
20
60
40
20
0 0
2
4
6
8
10
12
0 Acetonitrile
Ultrasonication time (min)
Methanol
Ethanol
Acetone NaOH solution
--
Eluent solvents
Figure 5. Effect of ultrasonication time. Extraction conditions: sample volume, 5.0 mL spiked with 200 ng/mL of sulpiride; pH 7.0; MNPs, 4 mg; eluent solvent, 0.2 mL of 0.02 mol/L NaOH.
accelerate the interactive rate between the MNPs and aqueous phase so that the target analytes could be well adsorbed on the surface of MNPs in a shorter time. The effect of ultrasonication time in the range of 0–10 min was evaluated at 25 Hz of ultrasonication frequency to reveal the effect of extraction time on the recovery of the sulpiride. As indicated in Fig. 5, the extraction recovery of the sulpiride reached a maximum at 4 min. Therefore, 4 min was chosen for the following experiments.
Effect of type and volume of eluent solvent In order to desorb the sulpiride from MNPs surfaces and obtain a high enrichment factor, a suitable eluent should be used and the sorbed analyte should be desorbed in a small volume. Various eluent solvents such as ethanol, acetonitrile, methanol, acetone and sodium hydroxide solution were investigated. As shown in Fig. 6, the highest extraction recovery of sulpiride was achieved using 0.02 mol/L NaOH solutions as the desorbing solvent, which could be explained by the sulpiride having a better solubility in NaOH solution than other solvents. The effect of eluent volume was also tested by eluting sulpiride with the range of 0.1–0.5 mL of NaOH. It was observed that the recovery of sulpiride was increased with increasing the volume NaOH from 0.1 to 0.5 mL. A 0.2 mL aliquot of 0.02 mol/L NaOH was used for the elution of the adsorbed sulpiride in subsequent experiments.
Figure 6. Effect of eluent solvent. Extraction conditions: sample volume, 5.0 mL spiked with 200 ng/mL of sulpiride; pH 7.0; MNPs, 4 mg; ultrasonication time, 4 min; eluent solvent, 0.2 mL of 0.02 mol/L NaOH.
Effect of other molecules The optimal experimental conditions described above were used to study the inhibitory effect of biological amines on the extractability of the proposed method, such as arginine, ascorbic acid, aspartic acid, L-valine, L-leucine and glycine. In addition, some other analog antipsychotic drugs including benzamide, tiapride, chlorpromazine hydrochloride and doxepin were added to the sample solutions. As shown in Table 1, an equimolar concentration of each compound had no significant influence on the adsorption of sulpiride in urine and blood samples, while at a higher molar excess (10:1) of these compounds, a slight decrease of adsorption efficiency was observed. This indicates that the selectivity of the adsorption of sulpiride to MNPs is very high.
Comparison with different MNPs The application of the proposed synthetic Fe3O4@SiO2 MNPs to extract the sulpiride from human urine and blood for HPLC/DAD analysis was compared with Fe3O4 MNPs and conventional HPLC/DAD analysis. Figure 7 shows a comparison of chromatograms of analysis of sulpiride in human urine and blood samples by HPLC/DAD (A) without pre-treatment, and by MSPE-HPLC/ DAD using Fe3O4 (B) or Fe3O4@SiO2 (C) as the extraction solvent. Magnetic nanoparticles have excellent extraction efficiency of sulpiride in biological fluids, and the extraction efficiency of
Table 1. Effect of other molecules on the adsorption of sulpiride from urine and blood samples Analyte
100 ng/mL sulpiride 100 ng/mL sulpiride 100 ng/mL sulpiride
Coexisting compounds
— 100 ng/mL biological aminesa 1000 ng/mL biological amines 100 ng/mL analog drugsb 1000 ng/mL analog drugs
Recovery (%)
RSD (%) (n = 6)
Urine
Blood
Urine
Blood
92.8 89.2 80.5 90.3 82.6
90.9 88.7 82.9 87.8 79.2
3.9 5.9 7.1 5.5 6.9
4.6 5.4 5.9 7.3 7.2
a
Biological components were arginine, ascorbic acid, aspartic acid, glycine, L-valine and L-leucine Analog drugs were benzamide, tiapride, doxepin and chlorpromazine hydrochloride.
b
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MSPE for determination of sulpiride in human urine and blood by HPLC mAU
A.HPLC/DAD
sulpiride from urine and blood. The reproducibility of the nanoparticles was described as the recovery of standard sulpiride added to human urine and blood. Then the intra-assay variation of the nanoparticles was measured, the RSD of which was
