In Situ Synthesis of Magnetic Multiwalled Carbon Nanotube

Mar 4, 2010 - Helmholtz Zentrum Muenchen, Institute of Ecological Chemistry, Department of BioGeoChemistry and Analytics, German Research Center for E...
1 downloads 5 Views 4MB Size
Download PDF
Anal. Chem. 2010, 82, 2743–2752

In Situ Synthesis of Magnetic Multiwalled Carbon Nanotube Composites for the Clean-up of (Fluoro)Quinolones from Human Plasma Prior to Ultrahigh Pressure Liquid Chromatography Analysis Gabriel Morales-Cid,† Agnes Fekete,‡ Bartolome´ M. Simonet,† Rainer Lehmann,§ Soledad Ca´rdenas,† Xianmin Zhang,| Miguel Valca´rcel,† and Philippe Schmitt-Kopplin*,‡ Helmholtz Zentrum Muenchen, Institute of Ecological Chemistry, Department of BioGeoChemistry and Analytics, German Research Center for Environmental Health, Ingolsta¨dter Landstraβe 1, D-85758 Neuherberg, Germany, Department of Analytical Chemistry, Marie Curie Building (Annex), Campus de Rabanales, University of Cordoba, E-14071 Cordoba, Spain, Division of Clinical Chemistry and Pathobiochemistry (Central Laboratory), Department of Internal Medicine 4, University Hospital of Tuebingen, D-72076 Tuebingen, Germany, and Department of Chemistry, Fudan University, No. 220, Handan Rd, Shanghai, China Magnetic nanoparticles (MNPs) were deposited onto multiwalled carbon nanotubes (MWCNTs) by in situ hightemperature decomposition of the magnetic precursor [iron(III)] and MWCNTs, in ethylene glycol. This one-step synthetic method was applied to commercially available carbon nanotubes (CNTs). Scanning electron micrographs of the resulting products revealed that MNPs decorated the surface of the MWCNTs. The hybrid nanoparticles thus obtained were used for sampling and cleanup in the determination of eight fluoroquinolones (FQs) and two quinolones (Qs) at trace levels by ultra performance liquid chromatography (UPLC). A systematic study of analyte adsorption and desorption was conducted with MNPs and MWCNTs separately. Although both solid phases adsorbed the analytes to some extent, the much higher recoveries were obtained by using the MNP-MWCNT composite which was thus selected to treat plasma samples containing FQs and Qs. Lower accuracies were determined at spiked plasma compared to the standard solution caused by the complexation affinity of the analytes with proteins because high recoveries were observed when deproteinization was performed before treating the sample with the magnetic MWCNTs. The performance characteristics of the optimized method were determined, and the method was applied to the analysis of plasma samples from antibiotic-treated patients. On the basis of the results, the use of an in situ synthesized MWCNTMNP composite allows the simple, expeditious sampling and treatment of such complex biological samples for the subsequent determination of FQs and Qs present at free form. * To whom correspondence should be addressed. E-mail: schmitt-kopplin@ helmholtz-muenchen.de. Fax: + 49 89 3187 3358. † University of Cordoba. ‡ German Research Center for Environmental Health. § University Hospital of Tuebingen. | Fudan University. 10.1021/ac902631h  2010 American Chemical Society Published on Web 03/04/2010

Magnetic carbon nanotube composites are hybrids of magnetite (Fe3O4) and/or maghemite (γ-Fe2O3) with single-walled (SWCNTs) or multiwalled carbon nanotubes (MWCNTs). These composites combine the unique optical, electrical, and mechanical properties of carbon nanotubes (CNTs), and the paramagnetic or ferromagnetic properties of magnetic nanoparticles (MNPs) at room temperature. Such advantages have enabled their use as tips for magnetic force microscopes,1 separators in wastewater treatment,2,3 biosensors,4,5 drug delivery systems,6 and biomanipulators.7 By contrast, their analytical potential has been the subject of little research that has focused primarily on the development of synthetic methods and characterization of the resulting products. Magnetic CNTs can be prepared in various ways. One way of obtaining magnetic CNT composites is by filling carbon nanotubes with iron oxide particles.8 This method, however, is restricted to large CNTs with an open end. One alternative, more widely applicable method involves attaching magnetic nanoparticles to the surface of nanotubes2,7,9-11 by exploiting the ability of the particles to decorate CNTs via a linking molecule such as pyrene11 (1) Deng, Z. F.; Yenilmez, E.; Leu, J.; Hoffman, J. E.; Straver, E. W. J.; Dai, H. J.; Moler, K. A. Appl. Phys. Lett. 2004, 85, 6263–6265. (2) Deng, Y. H.; Deng, C. H.; Yang, D.; Wang, C. C.; Fu, S. K.; Zhang, X. M. Chem. Commun. 2005, 5548–5550. (3) Jin, J.; Li, R.; Wang, H. L.; Chen, H. N.; Liang, K.; Ma, J. T. Chem. Commun. 2007, 386–388. (4) Hu, P.; Huang, C. Z.; Li, Y. F.; Ling, J.; Liu, Y. L.; Fei, L. R.; Xie, J. P. Anal. Chem. 2008, 80, 1819–1823. (5) Qu, S.; Huang, F.; Chen, G.; Yu, S.; Kong, J. Electrochem. Commun. 2007, 9, 2812–2816. (6) Tan, F. Y.; Fan, X. B.; Zhang, G. L.; Zhang, F. B. Mater. Lett. 2007, 61, 1805–1808. (7) Gao, C.; Li, W. W.; Morimoto, H.; Nagaoka, Y.; Maekawa, T. J. Phys. Chem. B 2006, 110, 7213–7220. (8) Korneva, G.; Ye, H. H.; Gogotsi, Y.; Halverson, D.; Friedman, G.; Bradley, J. C.; Kornev, K. G. Nano Lett. 2005, 5, 879–884. (9) Correa-Duarte, M. A.; Grzelczak, M.; Salgueirino-Maceira, V.; Giersig, M.; Liz-Marzan, L. M.; Farle, M.; Sierazdki, K.; Diaz, R. J. Phys. Chem. B 2005, 109, 19060–19063. (10) Li, W. W.; Gao, C.; Qian, H. F.; Ren, J. C.; Yan, D. Y. J. Mater. Chem. 2006, 16, 1852–1859.

Analytical Chemistry, Vol. 82, No. 7, April 1, 2010

2743

or by electrostatic self-assembling.2,7,9 These methods are applicable to a variety of CNTs, which, however, require some prior modification. In fact, the preparation method involves complicated, time-consuming operations, and coupling MNPs and CNTs in a controlled manner is frequently difficult.7 An alternative method involving the in situ synthesis of MNPs onto the surface of CNTs6,12-17 holds much promise for large-scale synthesis. Tan et al.6 prepared magnetic CNTs by dispersing nanotubes in iron pentacarbonyl for their vacuum thermolysis and oxidation. One other method for in situ preparation of MNP-CNT composites involves decomposing ferrocene at a high temperature (350-500 °C)15 onto previously purified CNTs. Chemical precipitation has also been used for in situ decoration of CNTs; the nanotubes are first oxidized in order to avoid breaking the tubes,11 and the process can be performed at fairly low temperatures.13 Gao et al. used chemical precipitation to decorate carbon nanotubes with iron oxide particles;17 the resulting hybrid particles spanned a broad range of sizes.12 Magnetic nanoparticles have also been produced onto the CNT surfaces by using a simple one-step hightemperature decomposition approach whereby the precursor (iron chloride or iron acetate) and MWCNTs are homogenized in ethylene glycol.12,14 Although a high temperature is needed for efficient decoration, the reaction is relatively selective and sensitive; also, the CNTs require no modification, and the size and coverage density of the MNPs can be readily adjusted via the MWCNT/magnetite precursor feed ratio, synthesis time, and temperature. In this work, we exploited the advantages of the solvothermal approach for decorating MWCNTs with MNPs to prepare magnetic CNT composites. The product was characterized by X-ray powder differentiation, transmission electron microscopy, Fourier transform infrared spectroscopy, and magnetic hysteresis analysis elsewhere;12,14 so, the present study focused on developing a straightforward decoration method to obtain composites of use in the targeted analysis of antibiotic traces in complex biological matrices such as human plasma. Fluoroquinolones (FQs) and quinolones (Qs) are broadspectrum antibiotic drugs. Ciprofloxaxin, the most widely used FQ at present, was introduced onto the clinical market in 1987. Sustained pharmaceutical research has led the Q family to grow to its fourth, current generation.18,19 Quinolones have become the most extensively used drugs for the treatment of bacterial infections in both human and veterinary medicine. Fluoroquinolones and quinolones differ in their mechanism of action; thus, Qs target the bacterial type II DNA topoisomerases,20 whereas FQs act by binding to gyrase or topoisomerase IV in the presence (11) Georgakilas, V.; Tzitzios, V.; Gournis, D.; Petridis, D. Chem. Mater. 2005, 17, 1613–1617. (12) Wan, J. Q.; Cai, W.; Feng, J. T.; Meng, X. X.; Liu, E. Z. J. Mater. Chem. 2007, 17, 1188–1192. (13) Kong, L. R.; Lu, X. F.; Zhang, W. J. J. Solid State Chem. 2008, 181, 628– 636. (14) Jia, B. P.; Gao, L. J. Phys. Chem. B 2007, 111, 5337–5343. (15) Sun, Z. Y.; Liu, Z. M.; Wang, Y.; Han, B. X.; Du, J. M.; Zhang, J. L. J. Mater. Chem. 2005, 15, 4497–4501. (16) Jiang, L. Q.; Gao, L. Chem. Mater. 2003, 15, 2848–2853. (17) Cao, H. Q.; Zhu, M. F.; Li, Y. G. J. Magn. Magn. Mater. 2006, 305, 321– 324. (18) Andriole, V. T. Clin. Infect. Dis. 2005, 41 (2), S113–119. (19) Oliphant, C. M.; Green, G. M. Am. Fam. Phys. 2002, 65, 455–464. (20) Marians, K. J.; Hiasa, H. J. Biol. Chem. 1997, 272, 9401–9409.

2744

Analytical Chemistry, Vol. 82, No. 7, April 1, 2010

of DNA.21 Various analytical methods based on spectrophotometry, mass spectrometry (MS), voltammetry, biosensing, and separation sciences (capillary electrophoresis, chromatography) are currently available for determining both compound families in matrices such as pharmaceutical, food, environmental, and biological samples; interested readers are referred to previous papers22-31 for further information. The high separation power and robustness of liquid chromatographic techniques including ultrahigh pressure liquid chromatography (UHPLC),32-34 reversedphase HPLC,35-38 and hydrophilic interaction liquid chromatography (HILIC)39 have been used for the separation of FQs and Qs in biological samples such as plasma,32,35-39 pig tissue,33 and milk34 although the sample treatment were less characterized. In this way, ciprofloxacin recoveries as high as 72% from human plasma have been obtained after centrifugation and C18 extraction.35 One other sample treatment used in this context involves protein precipitation with perchloric acid, vortex-mixing, and centrifugation; its proponents, however, focused on deproteination rather than on the development of a selective treatment for FQs.38 The purpose of this work was to assess the potential of a synthetic sorbent material with magnetic susceptibility resulting from the combination of MWCNTs and MNPs for target-selective sampling and sample preparation from complex matrices. The ensuing sample treatment was used in combination with UHPLC for the simultaneous determination of eight fluoroquinolones and two quinolones in human plasma. The results testify to the applicability of the proposed method to a variety of quinolone family members and generations. (21) Drlica, K.; Zhao, X. L. Microbiol. Molec. Biol. R 1997, 61, 377. (22) Okeri, H. A.; Arhewoh, I. M. Afr. J. Biotechnol. 2008, 7, 670–680. (23) Kaur, K.; Kumar, A.; Kumar, A.; Singh, M. B.; Rao, A. L. J. Crit. Rev. Anal. Chem. 2008, 38, 2–18. (24) Andreu, V.; Blasco, C.; Pico, Y. TrAC-Trends Anal. Chem. 2007, 26, 534– 556. (25) Samanidou, V. F.; Christodoulou, E. A.; Papadoyannis, I. N. Curr. Pharm. Anal. 2005, 1, 283–308. (26) Samanidou, V. F.; Christodoulou, E. A.; Papadoyannis, I. N. Curr. Pharm. Anal. 2005, 1, 155–193. (27) Hernandez, M.; Borrull, F.; Calull, M. TrAC-Trends Anal. Chem. 2003, 22, 416–427. (28) Hernandez-Arteseros, J. A.; Barbosa, J.; Compano, R.; Prat, M. D. J. Chromatogr. A 2002, 945, 1–24. (29) Belal, F.; Al-Majed, A. A.; Al-Obaid, A. M. Talanta 1999, 50, 765–786. (30) Carlucci, G. J. Chromatogr. A 1998, 812, 343–367. (31) Schmitt-Kopplin, P.; Burhenne, J.; Freitag, D.; Spiteller, M.; Kettrup, A. J. Chromatogr. A 1999, 837, 253–265. (32) Park, D. J.; Phapale, P. B.; Jang, I. J.; Cui, S.; Moon, B. J.; Kim, J. E.; Kim, W.; Hwang, S. K.; Yoon, Y. R. Chromatographia 2008, 68, 187–192. (33) Shao, B.; Jia, X.; Wu, Y.; Hu, J.; Tu, X.; Zhang, J. Rapid Commun. Mass Spectrom. 2007, 21, 3487–3496. (34) Stolker, A. A. M.; Rutgers, P.; Oosterink, E.; Lasaroms, J. J. P.; Peters, R. J. B.; van Rhijn, J. A.; Nielen, M. W. F. Anal. Bioanal. Chem. 2008, 391, 2309–2322. (35) Vybiralova, Z.; Nobilis, M.; Zoulova, J.; Kvetina, J.; Petr, P. J. Pharm. Biomed. Anal. 2005, 37, 851–858. (36) Liang, H. R.; Kays, M. B.; Sowinski, K. M. J. Chromatogr. B-Anal. Technol. Biomed. Life Sci. 2002, 772, 53–63. (37) Garcia, M. A.; Solans, C.; Calvo, A.; Royo, M.; Hernandez, E.; Rey, R.; Bregante, M. A. Chromatographia 2002, 55, 431–434. (38) Maya, M. T.; Goncalves, N. J.; Silva, N. B.; Morais, J. A. J. Chromatogr. B 2001, 755, 305–309. (39) Ji, H. Y.; Jeong, D. W.; Kim, Y. H.; Kim, H. H.; Sohn, D. R.; Lee, H. S. J. Pharm. Biomed. Anal. 2006, 41, 622–627.

EXPERIMENTAL SECTION Reagents and Standards. Both quinolones [Cinoxacin (CINO) and Nalidixic acid (NAL)] and the eight fluoroquinolones [Enoxacin (ENOX), Norfloxacin (NOR), Ofloxacin (OFLO), Ciproxacin (CIPRO), Danoflaxin (DANO), Lomeflaxin (LOME), Enrofloxacin (ENRO), and Difloxacin (DIFLO)] were purchased as ammonium hydroxide, sodium hydroxide, or sodium phosphate salts from Sigma Aldrich (Taufkirchen, Germany). The organic solvents used for separation (viz., acetonitrile, methanol, formic acid, and ammonium formate; all UPLC/MS grade) were supplied by Biosolve (Valkenswaard, The Netherlands). Water was obtained from a Milli-Q plus system (Millipore Corporation, Billerica, CA). Iron(III) chloride hexahydrate (Alfa Aesar, Karlsruhe, Germany), extra-pure sodium acetate (Sigma, Taufkirchen, Germany), and ethylene glycol (MERCK, Darmstadt, Germany) were used to synthesize the magnetic nanoparticles. Multiwalled carbon nanotubes 110-170 nm long and 5-9 µm thick (90% purity in MWCNTs) were purchased from MER (Tucson, AZ). Magnetic fields approximately 117.7 N in strength were applied by placing an axial magnetized disk (Ni-Cu-Ni) 20 mm in diameter and 10 mm thick with N42 magnetization (SuperMagnete, Zurich, Switzerland) on the outer wall of the glass vial. Standard stock solutions of the target analytes were prepared by dissolving the required amounts in methanol to obtain a 100 µg/mL concentration of each compound, and subsequently stored refrigerated at 4 °C. Human plasma samples from healthy and antibiotic-treated individuals were supplied by the University Hospital of Tuebingen (Germany). Samples were previously filtered through an Ultracel YM-3 Microcon Centrifugal Filter Device from Millipore (Bedford, MA), which uses regenerated cellulose of 3000 nominal molecular weight limit (NMWL) and a reservoir holding up to 0.5 mL of sample. Apparatus. UPLC analyses were performed by using an Acquity System instrument from Waters Corporation (Milford, MA) equipped with a 2996 PDA detector. The optimized configuration included an Acquity 2.1 × 150 mm BEH column packed with 1.7 µm C18 particles, also from Waters Corporation. The sample manager was thermostatted at 25 °C, and the column at 60 °C. A volume of sample of 10 µL was injected in the partial loop with needle overfill mode in each run. Detection was done at 279 nm for all analytes except NAL (λ ) 319 nm), using a scan rate of 80 Hz. The optimum mobile phase composition was 0.1% formic acid in water containing 10% acetonitrile (A) and 0.06% formic acid in acetonitrile (B). A linear solvent strength gradient was applied from 5 to 60% B within 5 min at a flow rate of 0.8 mL/min that caused the pressure to drop from 870 to 760 bar. The MWCNT-MNP composite was characterized using a Model 2000 FT-IR spectrophotometer from Perkin-Elmer (Waltham, MA) and a scanning electron microscope (SEM) from model JSM-6300F scanning electron microscope (Jeol, Munich, Germany) equipped with a field emission electron gun operated at 5 kV. pKa, log P, and log D were determined with the software Pallas 3.1 for Windows, using PrologP 7.0, pKalc 5.1, and PrologD 3.0 (CompuDrug, Budapest, Hungary) for the specific calculations. Preparation of the MWCNT-MNP Composite. An amount of 14 mg of FeCl3 · 6H2O and one of 4 mg of MWCNT were

suspended in 0.75 mL of ethylene glycol in a glass vial. Then, 0.036 g of sodium acetate was added and dissolved, and the solution allowed to stand at room temperature for 30 min, after which the glass vial was placed in an airtight steel container and heated in an oven at 200 °C for 16 h. After cooling to room temperature, the synthetic product was washed with 1 mL of water and nanoparticles were recovered by applying a magnetic field via a magnet placed on the outer wall of the glass vial. This cleanup procedure was repeated 5 times. The nanoparticles thus obtained can be stored in Milli-Q-water (1 mL) or dried at 80 °C until needed. Using the MWCNT-MNP Composite to Clean up Human Plasma Samples. An amount of 1.28 mg of MWCNT-MNP composite was accurately weighted inside a glass vial and supplied with 300 µL of EDTA plasma. Then, 700 µL of 10 mM formate buffer at pH 5.4 was added, and the mixture shaken for 1 min and allowed to stand for 10 min. The supernatant was then removed with a pipet and a magnet paced on the outer wall of the vial in order to collect the nanoparticles. Then, the vial and nanoparticles were both washed with 1 mL of 10 mM formate buffer at pH 5.4 that was subsequently removed with a pipet and the aid of the magnet, which was again placed on the outer wall of the vial. This was followed by elution of the analytes with 1 mL of methanol containing 10% ammonium hydroxide. After 1 min, nanoparticles were separated with the aid of the external magnet, and the supernatant was evaporated under a gentle N2 stream. Finally, the samples were recovered in 300 µL of 10 mM phosphate buffer at pH 8.2 for analysis by UPLC. RESULTS AND DISCUSSION UPLC Separation and UV-vis Detection. The lack of a method for simultaneously separating the studied FQs and Qs (Figure 1) led us to use ultrahigh pressure liquid chromatography (UHPLC) to this end. The UHPLC technique was used in combination with bridge-ethylene hybrid (BEH) and high-strength silica (HSS) as reversed-phase materials in order to facilitate the detection of differences in analyte retention. Surprisingly, not much difference was found, so BEH was adopted for further testing on the grounds of its increased pH tolerance. The peak widths obtained were less than 3 s wide and peaks symmetric (symmetry factor 0.8-1.3), which testifies to the suitability of the solid phase material for the target analytes. Because the analytes have pH-dependent cationic, anionic, and zwitterionic properties, their separation was performed at variable pH values. Above pH 7 (10 µmol/mL ammonium formate), peaks were asymmetric and exhibited some tailing due to inhomogeneous interaction. This led us to choose an acidified eluent (0.1% formic acid) for subsequent tests. Methanol was also tested as strong eluent in order to improve the selectivity. No improvement in resolution was obtained, however, which led us to adopt acetonitrile containing 0.6% formic acid on the grounds of its lower viscosity and UV cutoff. Also, the large differences in octanol-water partition coefficient between the analytes (0.11 to 2.39) required using gradient elution. A systematic study was performed in order to optimize the gradient composition and duration. The best results for the ten (fluoro)quinolones were obtained by using a gradient from 15% to 60% acetonitrile within 5 min; under these conditions, however, LOME and DANO failed to separate at baseline. Resolution of this critical pair of compounds was increased from Analytical Chemistry, Vol. 82, No. 7, April 1, 2010

2745

Figure 1. Structures of the studied FQs and Qs. Table 1. Analytical Figures of Merit of the Proposed UPLC/UV-vis Method for the Determination of Ten (Fluoro)quinolonesa analyte

retention time (min)

RSDb(%)

resolution

peak width (s)

RSDc(%)

slope

intercept

R2

LODd (ng/mL)

LOQe (ng/mL)

ENOX NOR OFLO CIPRO DANO LOME ENRO DIFLO CINO NAL

0.95 1.00 1.03 1.06 1.14 1.16 1.23 1.47 1.78 2.75

0.059 0.027 0.029 0.030 0.031 0.024 0.007 0.015 0.034 0.018

1.72 1.12 1.36 3.42 0.91 2.20 6.49 8.00 24.20

1.77 1.64 1.26 1.60 1.39 1.40 2.09 2.31 2.44 2.35

1.1 0.4 0.9 0.3 0.7 2.4 1.3 0.9 0.5 1.8

57.249 109.19 26.21 83.02 86.20 74.92 86.57 69.62 64.20 35.75

-291.90 -422.56 -173.67 -395.82 -629.13 -348.22 -1512.67 -705.92 -215.38 -174.88

0.9998 0.9997 0.9998 0.9998 0.9995 0.9997 0.9991 0.9991 0.9997 0.9998

5.8 7.1 6.1 6.1 9.0 7.4 14.5 12.0 6.9 5.9

19.2 23.4 20.4 20.3 30.0 24.7 48.2 39.9 23.1 19.6

a Calibration graphs were obtained at seven concentration levels. Separation column: Acquity BEH C18 (2.1 × 150 mm, 1.7 µm particle size). Sample manager temperature: 25 °C. Column temperature: 60 °C. Sample injection: 10 µL in the partial loop with needle overfill mode. Detection wavelength: 279 nm (319 nm for NAL). Mobile phase: 0.1% formic acid in water containing 10% acetonitrile (A) and 0.06% formic acid in acetonitrile (B). A linear solvent gradient was applied from 5 to 60% B within 5 min at a flow rate of 0.8 mL/min. b Within-day relative standard deviation of retention time (n ) 6). c Within-day relative standard deviation of peak area (n ) 6). d Limit of detection, calculated as 3Sy/x/slope. e Limit of quantitation, calculated as 10Sy/x/slope.

0.72 to 0.91 with no loss of separation efficiency by using a longer column, 150 mm, and higher scan rate which was therefore adopted for separation. In any case, resolution might be improved by using a selective detection technique such as MS, with which the proposed method is compatible. The performance characteristics of the optimized method, which are described in the Experimental Section, are summarized in Table 1. Retention times ranged from 0.95 to 2.75 min, and their repeatability, as RSD, ranged from 0.02 to 0.6. The separation efficiency was quite high; thus, peak widths in the dispersion zone ranged from 1.8 to 2.4 s. RSD for the peak area was less than 2.4% in all instances. A linear dependence (R2 > 0.999) was 2746

Analytical Chemistry, Vol. 82, No. 7, April 1, 2010

observed over the concentration range 15-300 ng/mL for all analytes. Limits of detection (LODs) spanned the range 5.77-14.46 ng/mL. Overall, the method proved quite selective, sensitive and fast for the ten (fluoro)quinolones. This encouraged us to use it for the development of a sampling-treatment procedure efficiently exploiting the advantages of the synthetic nanomaterials (namely, MNPs, MWCNTs, and MWCNT-MNP composites). Adsorption of (Fluoro)quinolones onto MNPs. Comparing adsorption and desorption of the analytes on the MWCNT-MNP composite entailed examining each component of the hybrid material individually since no information on the sorption proper-

Figure 2. Adsorption rate of quinolones (97 ng/mL) onto magnetic nanoparticles (640 mg/L) as a function of pH (3-9.3) after 1 min of contact time. Adsorption was determined by analyzing the free fraction of analytes present in the supernatant.

ties of the analytes on the MNPs or on the carbonaceous material were found. The initial tests involved studying adsorption and desorption on MNPs prepared as described elsewhere.40 The amount of analyte adsorbed on the magnetic particles was found to depend mainly on the following factors: (a) the pH of the solution (a result of the analytes being zwitterionic); (b) type of solvent (water or organic); (c) amount of magnetic nanoparticles (correlated to the amounts adsorbed); (d) analyte concentration, which should not exceed a given level in order to avoid saturation of the nanoparticles and the resulting breakthrough; (e) contact time, extension of which facilitates analyte-particle interactions until equilibrium is reached; and (f) amount of energy applied during the contact time (e.g., by shaking), which helps accelerate the adsorption kinetics. The pH of the solution had a critical effect on analyte-MNP interactions. Its influence was studied by placing MNPs at a concentration of 640 mg/L in contact with a standard solution containing the analytes at 97 ng/mL in a buffer at pH 3.0-9.3 for 1 min. As can be seen from Figure 2, adsorption, which was assessed from the concentrations of free analytes remaining in solution, peaked near pH 5.4. Such a pH, which was provided by 10 mM formate buffer, was adopted for subsequent adsorption tests. As can also been seen from Figure 2, Qs (CINO and NAL) exhibited lower adsorption rates on MNPs than did FQs. An alkaline pH provided by 1 mL of 10 mM phosphate buffer at pH 8.2 was the most effective elution solvent since the elution efficiency showed more roboust character in alkaline pH as in acidic condition as can be seen from Figure 2 (adsorption was near zero over the pH range 7.20-9.30 but substantial at 3.02-4.03). Using the recommended procedure and an analyte concentration of 25 ng/mL led to recoveries of 76-101%. Raising the analyte concentration to 50 ng/mL resulted in incomplete adsorption and, hence, in poor recoveries (34-55%, Table 3). The use organic eluents (hexane, isopropanol, acetonitrile, methanol, ethyl acetate, toluene, and methanol containing 10% ammonium hydroxide) was also studied. As can be seen from (40) Deng, H.; Li, X. L.; Peng, Q.; Wang, X.; Chen, J. P.; Li, Y. D. Angew. Chem., Int. Ed. 2005, 44, 2782–2785.

Table 2. Eluents Used to Recover the Analytes from MNPs and MWCNTsa MNPs

eluent hexane isopropanol acetonitrile methanol ethyl acetate toluene water, pH 8.2 methanol, 10 vol %, ammonium hydroxide

MWCNTs

number of number of analytes recoveries analytes recoveries detected (%) detected (%) 0 0 0 0 0 0 10 0

ndb nd nd nd nd nd 34.6-55.4 nd

0 10 10 10 3 5 1 10

nd 29-99 9.4-83 28.4-103 6.2-20.4 9.1-30.7 6.1 67.5-91.4

a The number of analytes detected with each eluent, and the maximum and minimum recoveries obtained, are also shown. b nd ) not detected.

Figure 3. Peak area as measured in the supernatant after preconcentration with variably concentrated MNPs in solution. Preconcentration medium: 10 mM formate buffer at pH 5.4. Analyte concentration: 50 mg/mL each.

Table 2, however, none of the organic solvents was capable of eluting the analytes from the MNPs. These results indicate that analyte-MNP interactions must be mostly of the polar or Analytical Chemistry, Vol. 82, No. 7, April 1, 2010

2747

Figure 4. Analyte recoveries obtained by using variable proportions of ammonium hydroxide in MeOH for elution after MWCNT based preconcentration. Preconcentration medium: 10 mM formate buffer at pH 5. Analyte concentration: 50 mg/mL each.

dipole-dipole type rather than ionic type since adsorption in the aqueous medium was maximal near the isoelectric point, and once the analytes were charged, their solubility in the mobile phase increased and their adsorption decreased as a result. The effect of the MNPs/analyte concentration ratio was also examined with a view to maximizing adsorption. Tests were performed in 1 mL volumes containing the FQs and Qs at a 50 ng/mL concentration each at pH 5.4 that were supplied with variable concentrations of MNPs from 640 to 2000 mg/L. As can be seen from Figure 3, MNP concentrations above 1280 mg/L failed to significantly increase analyte adsorption, so this particular concentration was chosen for further testing. The above-described optimum conditions were used to examine the adsorption kinetics of FQs and Qs on MNPs. Adsorption was found to peak after 30 min of contact time following 1 min of shaking. As expected, the adsorption kinetics was affected by shaking. In fact, a shaking time of 10 min proved long enough to ensure maximal adsorption. Adsorption of (Fluoro)quinolones onto MWCNTs. Our initial objective was to use the magnetic susceptibility of MNPs in order to simplify the sample treatment. However, as noted in the previous section, the sorption properties of the magnetic nanoparticles failed to ensure reliable sample preparation for the determination of FQs and Qs. This led us to test an alternative material capable of efficiently adsorbing aromatic compounds, namely: carbon nanotubes (CNTs), which can also be used in combination with MNPs. We chose to use MWCNT in preference to SWCNT in order to obtain an effective hybrid material (a composite) as regards adsorption. This choice rested on three major arguments, namely: (a) MWCNTs possess a higher adsorption capacity than SWCNTs;41 (b) MWCNTs are less prone to aggregating;42 and (c) MWCNTs are more widely used for SPE than are SWCNTs.43 Tests were performed with three different types of MWCNTs purchased in purity greater than 90% from (41) Valcarcel, M.; Cardenas, S.; Simonet, B. M. Anal. Chem. 2007, 79, 4788– 4797. (42) Dai, L. Carbon Nanotechnology: Recent Developments in Chemistry, Physics, Material Science and Device Applications; Elsevier: Amsterdam, 2006. (43) Valcarcel, M.; Cardenas, S.; Simonet, B. M.; Moliner-Martinez, Y.; Lucena, R. TrAC-Trends Anal. Chem. 2008, 27, 34–43.

2748

Analytical Chemistry, Vol. 82, No. 7, April 1, 2010

Table 3. Recoveries and RSD Values Obtained in the Analysis of Ten (Fluoro)quinolones in a Standard Water Solution by Using the Optimized Sample Clean-up Method with Three Types of Nanoparticlesa recovery (%) and RSD (%) analyte

MNPs

MWCNTs

MWCNT-MNP

ENOX NOR OFLO CIPRO DANO LOME ENRO DIFLO CINO NAL

41.7 ± 4.7 42.0 ± 5.7 43.8 ± 4.4 44.9 ± 4.7 37.1 ± 7.9 45.4 ± 3.0 52.6 ± 5.8 50.4 ± 3.3 34.6 ± 0.6 55.4 ± 2.6

69.7 ± 2.3 70.0 ± 1.1 77.3 ± 5.4 77.4 ± 1.2 71.0 ± 8.1 80.0 ± 1.1 67.5 ± 8.5 74.3 ± 2.8 91.4 ± 0.3 89.1 ± 0.3

95.3 ± 2.4 99.3 ± 2.1 83.3 ± 3.8 101.3 ± 3.2 102.4 ± 5.5 94.2 ± 1.8 75.6 ± 5.7 81.9 ± 2.9 81.8 ± 0.5 78.8 ± 0.7

a Each analyte was added at a concentration of 50 ng/mL, and each sorbent used in six analyses (n ) 6).

Bayer (Leverkusen, Germany), MER (Tucson, AZ, USA) and NTP (Shenzhen, China); their respective nanotube dimensions were 5-20 nm × 1-10 µm, 110-170 nm × 5-9 µm, and 10-30 nm × 5-15 µm. The experimental conditions for these adsorption tests (amount of material, type and volume of liquid phase, and shaking and contact times) were the same as those previously optimized for the MNPs since their use in hybrid form was planned at a later stage. By exception, the analyte concentrations were raised to 50 ng/mL in order exploit the expected increased adsorption capacity of MWCNTs relative to MNPs. Since MWCNTs exhibit no magnetic susceptibility, use of the magnetic field to separate particles from the supernatant was replaced with centrifugation at 14 000 rpm for 7 min. After preconcentration, which involved 1 min of shaking and 10 min of contact time, UPLC analysis of the supernatant from the solutions containing MER and NTP MWCNTs revealed the absence of analytessat least at detectable concentrations. Complete adsorption of the analytes was therefore assumed. On the other hand, adsorption on the Bayer MWCNTs was incomplete since the supernatant contained traces of all analytes. This may have resulted from the stronger tendency of these nanotubes to aggregatesin fact, they are supplied as pellets. Subsequent tests were therefore performed with MER MWCNTs,

Figure 5. SEM images of the nanoparticles at the micrometric scale and detail at the 500 nm scale for each type of nanomaterial: (a) Magnetic manoparticles (MNPs); (b) multiwalled carbon nanotubes (MWCNTs); (c) MWCNT-MNP composite. All samples were coated with a platinum film.

which were the only nanotubes ensuring thorough adsorption of the studied analytes. Elution was studied with various solvents. On the basis of the results obtained with aqueous solutions at pH 3-9.3, only CINO was eluted (at pH 8.2, Table 2). Several organic solvents (viz. hexane, acetonitrile, methanol, ethyl acetate, and toluene) were also tested. The best results were provided by methanol, which, however, resulted in incomplete recovery of many analytess recoveries ranged from 28.4 to 103% (Table 2). Since the analytes were more soluble in the organic solvents than in the water used as mobile phase, their interaction with MER MWCNTs must be of the nonpolar or π-π type. The feasibility of improving the elution efficiency of methanol was examined by adding formic acid or ammonium hydroxide to adjust its apparent pH. Using formic acid or ammonium hydroxide, both in a 1% proportion, increased the recoveries to 31-89% and 36-93%, respectively, which led us to adopt the former as modifier as shown in Figure

4. As can be seen, the recoveries for most of the analytes peaked at 70-91% with a 10% concentration of ammonium hydroxide in methanol and no further improvement was observed by raising it to 15%. Therefore, a 10% concentration of modifier was selected as optimal (Table 3). Preparation and Characterization of the MWCNT-MNP Composite. The aim here was to obtain a hybrid nanomaterial combining the magnetic properties of MNPs at room temperature and the extremely high sorption capacity of MWCNTs by using the thermal decomposition approach, which provides an easy, relatively fast, one-step synthetic procedure. This advantage could therefore be used for the convenient, effective sampling and clean-up of the target components in a single step, even from a complex matrix. The MWCNT-MNP composite was prepared using the same procedure as in the synthesis of MNPs except for the addition of MWCNTs swhich were intended to provide a core for MNP formationsin the first step.12 The Analytical Chemistry, Vol. 82, No. 7, April 1, 2010

2749

Figure 6. Chromatograms for various types of samples as obtained by using UPLC in combination with UV-vis detection at 279 nm for 2.5 min and 319 nm for 2.5 min. (A) 100 ng/mL standard solution of the quinolone mixture. (B) Plasma sample spiked with a 100 ng/mL concentration of each analyte and subjected to the MWCNT-MNP based procedure. (C) Unspiked plasma sample subjected to the same treatment as B (MWCNT-MNP) and testing positive for OFLO and CIPRO. Peaks: (1) ENOX, (2) NOR, (3) OFLO, (4) CIPRO, (5) DANO, (6) LOME, (7) ENRO, (8) DIFLO, (9) CINO, (10) NAL. 2750

Analytical Chemistry, Vol. 82, No. 7, April 1, 2010

amount of MWCNTs used was that needed to obtain an MWCNT-MNP weight ratio 1:1, and thus, it was possible to utilize the sorption property of both material when they were present as a composite. The amount of MNPs previously obtained by using the same reagent concentration and working conditions except for the absence of MWCNTs was 4 mg. A yield of 62.28% as calculated by dividing the amount of product obtained, in grams, into the sum of MWCNT and FeCl3 · 6H2O added, also in grams, was obtained. The nanomaterial thus obtained was rapidly isolated from the dispersion with the aid of an external magnetic field (117.7 N). No free MWCNTs remained in solution after the magnetic field was applied to the side wall of the vessel. This result is especially interesting for analytical purposes. The composite material could be easily dispersed, is stable, and settles at a low rate. In fact, it remained in suspension throughout the preconcentration process. In order to confirm whether that product obtained was in fact the MWCNT-MNP composite, the material was characterized by Fourier transform infrared spectroscopy (FTIR) and scanning electron microscopy (SEM). The FTIR spectrum for MNPs exhibits a typical band for Fe3O4 at 555 cm-1 that was absent from that for the composite. No spectral differences between MWCNT and the MWCNT-MNP composite were observed, however; this reflects the dominance of the properties of CNTs in the hybrid material, which, however, exhibits magnetic properties not present in the pure MWCNTs. Figure 5 shows SEM images for the three types of nanoparticles studied. Some differences are immediately apparent. Thus, the micrographs for the MNPs reveal formation of nanosphere aggregates ca. 100-200 nm in diameter (Figure 5A). Figure 5B shows a micrograph for the MWCNTs together with a detail of the MER MWCNTs at the 500 nm scale. Finally, Figure 5C is a micrograph of the composite material (MWCNT-MNP). The detailed views (500 nm scale) clearly reveal that the MNPs attach onto nanotube surfaces and look like nodes growing from the tubes. MNP-CNT binding in the composite is strong enough to resist applied mechanical energy such as that of manual shaking or sonication. The overall tubular structure of the MWCNTs remains intact after in situ decoration. The amount of MNPs used clearly appropriate, as reflected in absence of a well-defined morphology in the MNP nanoclusters and free MWCNTs. As can be seen, the MNPs obtained in isolation were smaller. This can be ascribed to the presence of MWCNT reducing their size. Because the nanotubes were not completely covered, the composite was expected to possess the sorption properties of both MNPs and MWCNTs that was aimed in the study. The sorption properties of the MWCNT-MNP composite for the eight FQs and two Qs were examined by using a 50 ng/ mL concentration of each analyte. Tests were performed with the same adsorption medium (10 mM formate buffer at pH 5.4) and eluent as in the previously optimized preconcentration procedures for MNP and MWCNT (viz. 10 mM phosphate buffer at pH 8.2 and methanol containing 10% ammonium hydroxide, respectively). The recoveries thus obtained ranged from 17.2 to 39.0% with the aqueous eluent and from 76 to 102% with methanol containing 10% of ammonium hydroxide. The latter eluent was therefore chosen to preconcentrate the

Table 4. Recoveries and RSD Values Obtained in the Analysis of Ten (Fluoro)quinolones in a Spiked Sample at a 100 ng/mL Concentration Each to Unfiltered and Filtered Human Plasma Samples by Using the Proposed MWCNT-MNP Based Clean-up and UPLC Separation Methoda unfiltered samples b

filtered samples

analyte

recovery (%)

RSD (%)

recovery (%)

RSD (%)

ENOX NOR OFLO CIPRO DANO LOME ENRO DIFLO CINO NAL

54.9 49.2 31.8 35.3 70.7 29.6 59.9 32.0 ndc nd

1.7 3.1 0.6 2.8 4.0 1.9 5.6 2.8

94.6 98.7 95.8 100.2 88.4 89.3 97.1 74.3 70.4 76.5

2.3 4.5 2.7 3.0 3.2 1.4 4.8 2.9 3.8 3.9

a Filtration was done with a centrifugal device containing regenerated cellulose with a 3000 nominal molecular weight limit (NMWL). Centrifugation program: 13 500 rpm for 30 min. b RSDs were calculated from six measurements of each sample (n ) 6). c nd ) not detected.

composite as shown in Table 3. The recoveries applying the composite for the treatment of the analytes in standard solution were better than those obtained with MNPs and MWCNTs that suggests that both materials contribute similarly to the adsorption characteristics of the composite. Also, the magnetic MWCNTs exhibited a lower sedimentation rate and less marked aggregation due to the net-like structure (Figure 5C). Additionally, the MWCNT-MNP hybrid was efficiently regenerated with the eluent (methanol containing 10% ammonium hydroxide) and reused with no loss of sorption capacity at least five times. Using the MWCNT-MNP Composite for Sample Treatment in the Determination of Free (Fluoro)quinolones in Human Plasma. The potential of the composite material for application to complex samples was examined in the determination of FQs in human plasma samples not subjected to the deproteinazation prior to clean-up to study the matrix effect. Figure 6 shows three chromatograms obtained by using the proposed MWCNTMNP clean-up method in combination with UPLC. As can be seen, all FQs were detected in the spiked plasma, but the analyte recoveries ranged from 31.8 to 70.7% (Table 4) and quinolones (CINO and NAL) went undetected as the likely result of their completely binding to proteins in plasma. Finally, Figure 6C shows the chromatogram for an unspiked plasma sample from a patient under treatment with fluoroquinolones (Ciprobay, mainly, in addition to other antibiotics). As can be seen, OFLO and CIPRO were detected with improved accuracy: the former was found to be present at 25.53 ng/mL and the latter at 98.67 ng/mL. A filtration step allowing the removal of proteins was introduced in order to eliminate the matrix effect and thus improve recoveries of the FQs and Qs. To this end, spiked human plasma samples were passed through a centrifugal filtering device containing regenerated cellulose with a nominal molecular weight limit (NMWL) of 3000. Centrifugation was done at 13 500 rpm for 30 min. As can be seen from Table 4, filtration resulted in substantially increased recoveries for all analytes. Also, both Qs Analytical Chemistry, Vol. 82, No. 7, April 1, 2010

2751

Figure 7. Chromatogram obtained by using the MWCNT-MNP composite based method to analyze plasma samples filtered with a centrifugal device containing regenerated cellulose of 3000 nominal molecular weight limit (NMWL). Centrifugation was done at 13 500 rpm for 30 min. Peaks: (1) ENOX, (2) NOR, (3) OFLO, (4) CIPRO, (5) DANO, (6) LOME, (7) ENRO, (8) DIFLO, (9) CINO, (10) NAL.

were recovered by more than 70% (Figure 7). On the basis of these results, the poorer recoveries obtained from unfiltered samples can be ascribed to competition between the sample proteins and the MWCNT-MNP composite for binding to the analytes. CONCLUSION A MWCNT-MNP composite was efficiently prepared by using a one-step in situ synthetic method. Deposition of MNPs onto the surface of MWCNTs endowed the product with magnetic properties. Using a sorbent phase with magnetic susceptibility has the advantage that it facilitates separation of the solid material from the solution by means of an externally applied magnetic field. This can considerably simplify sample preparation procedures. The composite material was found to provide improved mean recoveries (88.9%) of antibiotics of the quinolone family from human plasma relative to the use of MNPs (44.4%) or MWCNTs (76.4%) in isolation. The ensuing sampling and sample treatment method was successfully used to determine FQs in plasma from antibiotic-treated patients following fast UPLC separation of the analytes (only 3 min per

2752

Analytical Chemistry, Vol. 82, No. 7, April 1, 2010

analysis). As an added value, the proposed method can be transferred, with some reoptimization, to other separation techniques and detector types. ACKNOWLEDGMENT G.M.-C. gratefully acknowledges funding by Spain’s Ministry of Innovation and Science of his stay in Germany at the Helmholtz Zentrum Muenchen for 3 months to conduct the research work reported in this paper. The work was partially supported by the Kompetenznetz Diabetes mellitus (Competence Network for Diabetes mellitus) and funded by the Federal Ministry of Education and Research (FKZ 01GI0803-04) and the Sino-German Center for Research Promotion (DFG and NSFC, GZ 364). We thank Helga Wehnes and the Institute of Pathology at Helmholtz Zentrum Muenchen for allowing us to make measurements with the scanning electron microscope.

Received for review November 17, 2009. Accepted February 11, 2010. AC902631H