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Online In-Tube Solid-Phase Extraction Using a Nonfunctionalized PVC Tube Coupled with ICPMS for in Vivo Monitoring of Trace Metals in Rat Brain Microdialysates C. K. Su,† T. W. Li,† and Y. C. Sun*,†,‡ Department of Biomedical Engineering and Environmental Sciences and Nuclear Science and Technology Development Center, National Tsing-Hua University, 30013 Hsinchu, Taiwan We have developed a simple, automatic, online in-tube solid-phase extraction (SPE) method for the preconcentration of trace elements from saline samples. The method takes advantage of the adsorption of trace metal ions onto the interior of a nonfunctionalized PVC tube as a means of separation from the matrix salt. The adsorption of transition metal ions is presumably dominated by Lewis acid/base interactions, which facilitate the formation of metal-PVC complexes. For simultaneous determination of multiple trace metals in the extracellular fluid from the brains of anesthetized rats, we developed an online analytical system comprising microdialysis sampling, the established in-tube extraction procedure, and ICPMS. In the extraction step, the efficiency was optimal when the pH of the sample was adjusted to 8.0 using phosphate buffer. After extraction onto the interior of PVC tube, the adsorbed analytes were eluted with 0.5% HNO3 prior to online ICPMS measurement. For those elements present in the extracellular fluid at less than nanogram-permilliliter concentrations (i.e., Cu, Zn, and Mn), a temporal resolution of 12 min was required to collect enough microdialysate to meet the sensitivity requirements of the ICPMS instrument. Noteworthily, because washing and postconditioning the interior of PVC tube are not necessary, a relatively unsophisticated and clean procedure was attained and extremely low detection limits in the range of 5.0 to 510 ng L-1 were thus obtained for the analysis of Mn, Cu, Zn, Pb, Cd, Ni, and Co in microdialysate samples of 8 µL by ICPMS. To the best of our knowledge, this study is the first to exploit PVC peristaltic tubing as an SPE adsorbent in the laboratory-on-valve mode for online sample treatment and trace metal preconcentration prior to ICPMS measurement. We confirmed the analytical reliability of this method through the analysis of the certified reference material SLEW-3 (estuarine water) and 2670a (human urine) and demonstrated its applicability through simultaneous in vivo monitoring of the basal concentrations and concentration profiles of multiple metal ions in the brain extracellular fluid of a living rat. * To whom correspondence should be addressed. Phone:886-3-5727309. Fax: +886-3-5723883. E-mail:
[email protected]. † Department of Biomedical Engineering and Environmental Sciences. ‡ Nuclear Science and Technology Development Center. 10.1021/ac800802e CCC: $40.75 2008 American Chemical Society Published on Web 07/29/2008
In the brain, one of the main functions of the extracellular fluid and neurons is to convey messages as rapidly and as selectively as possible. To satisfy the requirement described above, unimpeded diffusion of messengers, such as metal ions and organic neurotransmitters, in the extracellular space is considered as one of the most vital factors.1 In spite of trace elements being minor building components in the nervous system, they are essential for many metabolic processes, and their homeostasis has been proven to be crucial to normal brain function. To date, there is much evidence showing that aberrations in cellular metal ion concentration may cause cell death and various neurological diseases, such as Alzheimer’s disease, Parkinson’s disease, Wilson’s disease and so on.2 Besides chronic neurodegenerative disorders, physical stresses, such as stroke or injury, can also induce changes in blood-brain barrier permeability3 and neural activity,4 that may also cause aberrations in brain metal concentrations. Based on research during the past several decades, dyshomeostasis in the concentrations of brain zinc,5 cobalt,6 lead,7 copper,8 manganese,9 cadmium,10 and nickel11 has been considered as a risk factor for neurological disorders. However, according to Thompson et al.,12 direct evidence is still lacking for the elucidation of the release and uptake of metal ions during neurological disorders. Because those commonly used analytical methods all cannot determine trace metal ions, nor their kinetics and spatial distribution with much resolution, up to now, the linkage between barrier dysfunction and the etiology of various neurological disorders remains unclear and the exploration of physiological roles of various trace metal ions involved in many (1) Williams, R. J. P. Inorg. Chim. Acta 2003, 356, 27–40. (2) Sigel, A.; Sigel, H.; Sigel, R. K. O. Metal Ions in Life Sciences; John Wiley & Sons: Chichester, U.K., 2006. (3) Nelson, N. EMBO J. 1999, 18, 4361–4371. (4) Choi, D W.; Koh, J. Y. Annu. Rev. Neurosci. 1998, 21, 347–75. (5) Konoha, K.; Sadakane, Y.; Kawahara, M., J. Health Sci. 2006, 52, 1–8. (6) Olivier, G.; Hess, C.; Savaskan, E.; Ly, C.; Meier, F.; Baysang, G.; Brockhaus, M.; Muller-Spahn, F. J. Pineal Res. 2001, 31, 320–325. (7) Garza, A.; Vega, R.; Soto, E. Med. Sci. Mon. 2006, 12, RA57–RA65. (8) Wimalasena, D. S.; Wiese, T. J.; Wimalasena, K. J. Neurochem. 2007, 101, 313–326. (9) Reaney, S. H.; Bench, G.; Smith, D. R. Toxicol. Sci. 2006, 93, 114–124. (10) Mendez-Armenta, M.; Rios, C. Environ. Toxicol. Pharm. 2007, 23, 350– 358. (11) Slotkin, T. A.; MacKillop, E. A.; Ryde, I. T.; Tate, C. A.; Seidler, F. J. Environ. Health Perspect. 2007, 115, 93–101. (12) Thompson, R. B.; Whetsell, W. O.; Maliwal, B. P.; Fierke, C. A.; Frederickson, C. J. J. Neurosci. Meth. 2000, 96, 35–45.
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different behaviors also remains as a demanding challenge for the development of neuroscience.13 In the CNS, except for copper and zinc, the role of trace metal ions in neural functions is poorly understood because of their low concentrations in the brain. According to Delgado et al.,14 not only are the extracellular concentrations of metal ions at very low levels but their non-protein-bound concentrations in synaptic space are much lower, in the range of sub-µg L-1 to µg L-1 for Zn and Mn, since they are probably bound to proteins. In vivo monitoring of the dynamic variations in the concentrations of trace metal ions in extracellular fluid is an invaluable tool for elucidating the release of metal ions from neurons and the extravasation of metal ions from the blood-brain barrier (BBB) during neurological disorders such as ischemia and reperfusion, posttraumatic disorder, and epilepsy.15,16 Despite continuing progress in improving the detectability of instrumental methods of analysis, the direct determination of trace metals in small volume biological samples remains difficult because of insufficient sensitivity or selectivity. In rat brain microdialysates, the combination of extraordinarily low concentrations of analyte cations (0.01-10 µg L-1)17–22 and the extremely complicated salt matrixsca. 0.9% of dissolved solidssoften makes the determination of trace metals difficult and imposes great demands on the capabilities of the instrumentation and techniques. Although ICPMS is a very powerful technique for the analyses of trace and isotopic samples, interference from high levels of alkali and alkaline earth metals causes molecular ion and plasma equilibrium shifts to occur, which can often lead to significant spectral interference and/or loss of sensitivity during trace metal determination.23 In addition, severe error can also occur as a result of salt buildup on the cone tipscaused by the introduction of large amounts of sodium in the matrixsduring prolonged measurement times. Thus, long-term, continuous in vivo monitoring of trace metals in the brains of anesthetized rats requires chemical separation to isolate the analyte ions of interest and to remove most of the interfering constituents prior to ICPMS analysis. Flow injection (FI) manifolds incorporating various online sample pretreatment and detection systems are widely employed to improve the accuracy and reproducibility of trace analysis24–26 because samples being analyzed in a closed system are less susceptible to contamination and are subjected to unchanging analytic conditions or reduced operator error relative to those (13) Harris, C. M. Anal. Chem. 2001, 73, 590 A. (14) Delgado, R.; Vergara, C.; Wolff, D. Biol. Res. 2006, 39, 173–182. (15) Kaufer, D.; Friedman, A.; Seidman, S.; Soreq, H. Nature 1998, 393, 373– 377. (16) Angel, I.; Bar, A.; Horovitz, T.; Taler, G.; Krakovsky, M.; Resnitsky, D.; Rosenberg, G.; Striem, S.; Friedman, J. E.; Kozak, A. Drug Dev. Res. 2002, 56, 300–309. (17) Itoh, T.; Saito, T.; Fujimura, M.; Watanabe, S.; Saito, K. Brain Res. 1993, 618, 318–322. (18) Itoh, T.; Saito, T.; Watanabe, S.; Saito, K. Trace Elem. Electrolytes 1996, 13, 196–199. (19) Tseng, W. C.; Sun, Y. C.; Yang, M. H.; Chen, T. P.; Lin, T. H.; Huang, Y. L. J. Anal. At. Spectrom. 2003, 18, 38–43. (20) Takeda, A.; Sotogaku, N.; Oku, N. Brain Res. 2003, 965, 279–282. (21) Chung, Y. T.; Ling, Y. C.; Yang, C. S.; Sun, Y. C.; Lee, P. L.; Lin, C. Y.; Hong, C. C.; Yang, M. H. Anal. Chem. 2007, 79, 8900–8910. (22) Sun, Y. C.; Lu, Y. W.; Chung, Y. T. J. Anal. At. Spectrom. 2007, 22, 77–83. (23) Chang, Y. L.; Jiang, S. J. J. Anal. At. Spectrom. 2001, 16, 1434–1438. (24) Miro´, M.; Frenzel, W. Microchim. Acta 2004, 148, 1–20. (25) Hansen, E. H.; Wang, J. W. Anal. Chim. Acta 2002, 467, 3–12. (26) Long, X. B.; Miro´, M.; Hansen, E. H. Anal. Chem. 2005, 77, 6032–6040.
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occurring during manual analysis or interpretation of results. To develop a sophisticated online analytical system for the determination of trace metals in saline samples, several attempts have been made to combine FI systems with sample pretreatment methods for the preconcentration of analytes prior to instrumental detection. The most frequently encountered online preconcentration procedures for trace elements include solvent extraction,27,28 solid-phase microcolumn extraction,29,30 precipitation/coprecipitation,31,32 and filterless preconcentration (knotted reactor).33,34 Among these preconcentration procedures, solid-phase extraction (SPE) methods are generally superior in terms of their simplicity, rapidity, and ability to provide high enrichment factors. For sorption preconcentration, solid sorbents such as activated carbon,35 porous organic polymers,36 functionalized polymeric sorbents,37 ion exchange resins,38 chelating resins presenting selective functional groups covalently attached to copolymer matrices,39 and ligand-modified resins40 have all been applied to the preconcentration of trace metals and metalloids from aqueous solutions. During the past decade, Fang et al. developed a unique online preconcentration system based on the retention of soluble metal complexes or precipitates on the inner walls of a knotted reactor (KR) prepared using PTFE tubing.41 Because of its simplicity and high cost-effectiveness, this novel procedure has been successfully and widely applied to trace element determination in biological and environmental samples.42,43 SPE methods can be categorized into two types: those that retain metal ions without the need to add any complexing reagent and those that require the addition of a complexing reagent to convert the analyte ions into extractable species prior to extraction. During the determination of trace metals, especially at small volumes of microdialysate (ca. 10 µL), the level of the method blank usually dictates the detectability and applicability of the analytical methods; thus, care must be taken to minimize the degree of reagent contamination when using a complexing reagent. For packed column systems, the presence of a tremendous amount of salt matrix and the pH-dependent affinity of the sorbent toward the analyte ions mean that the extraction efficiency achieved when using a functionalized resin (e.g., Chelex-100) (27) Saad, B.; Chong, C. C.; Ali, A. S. M.; Bari, M. F.; Ab Rahman, I.; Mohamad, N.; Saleh, M. I. Anal. Chim. Acta 2006, 555, 146–156. (28) Kara, D.; Fisher, A.; Hill, S. J. Analyst 2005, 130, 1518–1523. (29) Wang, J. H.; Hansen, E. H. J. Anal. At. Spectrom. 2001, 16, 1349–1355. (30) Hirata, S.; Kajiya, T.; Aihara, M.; Honda, K.; Shikino, O. Talanta 2002, 58, 1185–1194. (31) Tang, X. D.; Xu, Z. R.; Wang, J. H. Spectrochim. Acta B 2005, 60, 1580– 1585. (32) Andersen, J. E. T. Analyst 2005, 130, 385–390. (33) Benkhedda, K.; Dimitrova, B.; Infante, H. G.; Ivanova, E.; Adams, F. C. J. Anal. At. Spectrom. 2003, 18, 1019–1025. (34) Benkhedda, K.; Infante, H. G.; Ivanova, E.; Adams, F. C. J. Anal. At. Spectrom. 2001, 16, 995–1001. (35) Cerutti, S.; Silva, M. F.; Gasquez, R. A.; Olsina, R. A.; Martinez, L. D. Spectrochim. Acta B 2003, 58, 43–50. (36) Narin, I.; Soylak, M.; Elci, L.; Dogan, M. Anal. Lett. 2001, 34, 1935–1947. (37) Moawed, E. A.; Zaid, M. A. A.; El-Shahat, M. F. Anal. Lett. 2003, 36, 405– 422. (38) Sun, Y. C.; Tu, Y. L.; Mierzwa, J. Fresenius’ J. Anal. Chem. 1998, 360, 550–555. (39) Geckeler, K. E. Pure Appl. Chem. 2001, 73, 129–136. (40) Kilian, K.; Pyrzyn ˜ska, K. Fresenius’ J. Anal. Chem. 2001, 371, 1076–1078. (41) Fang, Z.; Xu, S.; Dong, Z. L.; Li, W. Talanta 1994, 41, 2165–2172. (42) Yan, X. P.; Jiang, Y. Trends Anal. Chem. 2001, 20, 552–562. (43) Cerutti, S.; Martinez, L. D.; Wuilloud, R. G. Appl. Spectrosc. Rev. 2005, 40, 71–101.
Figure 1. Schematic representation of the flow injection laboratory-on-valve system for trace element preconcentration via complexation with a nonfunctionalized PVC tube. VA and VC: six-port rotary valves. VB: eight-port rotary valves.
might be suppressed and that large volumes of washing and conditioning solutions might be needed to remove the extracted matrix ions and convert the resin into a form suitable for separation.38 Similarly, for KR systems,43 the need of adding complexing or precipitating reagents might also prohibit its accommodation of promoting the enrichment capability and detecting easily contaminated elements, such as Pb, Zn and Cu. To improve the practicability and effectiveness of SPE methods for the separation of trace elements in microdialysate samples, there is still high demand for simpler procedures and lower-blank methods. For online separation of trace elements from microdialysate samples, it is essential that the sorbent exhibit (i) high distribution coefficients for the analytes, but not for the salt matrix, (ii) appropriate capacity, and (iii) fast kinetics of sorption and elution. Recently, Eboatu et al.44 employed pure poly(vinyl chloride) (PVC) as a sorbent to separate transition metal ions from aqueous solutions by means of Lewis acid/base interactions. Meanwhile, Watts et al.45 reported that the adsorption efficiency of sodium ions is extremely low on PVC because it behaves as an acidic polymer. Because the use of open tubular reactors, such as KRs, has proven to be a superb approach toward eliminating the drawbacks associated with flow resistance during SPE, we suspected that a nonfunctionalized PVC tube/peristaltic pump tube would allow online preconcentration of trace elements in samples featuring a high salt content. To the best of our knowledge, nonfunctionalized PVC has never been incorporated into an online separation system for the SPE of trace metals, even though it might lead to simpler and cleaner online separation systems exhibiting sample enrichment and separation capability. Stable and reliable long-term ICPMS measurement of trace metal ions in microdialysates requires removal of the salt matrix and enhancement of the analyte signals. This paper describes a hyphenated system for the selective, sensitive, and continuous (44) Eboatu, A. N.; Diete-Spiff, S. T.; Ezenweke, L. O.; Omalu, E. J. Appl. Polym. Sci. 2002, 85, 2781–2786. (45) Watts, J. F.; Chehimi, M. M. J. Adhes. 1993, 41, 81–91.
determination of trace metals in microdialysates; it employs a separation process exploiting the complexation of pure metal ions with nonfunctionalized PVC, in conjunction with online ICPMS determination. The unique and highly selective Lewis acid/base interactions between nonfunctionalized PVC and metal ions does not require the addition of either chelating or (co)precipitating reagents, nor does it require cleaning or postconditioning procedures, to extract desired transition metal ions from saline solutions. We optimized the metal-polymer complexation process for the sorption preconcentration of trace metals and attempted to understand the selectivity of PVC toward metal ions. After optimizing the operating conditions for the online separation process, we arranged a Micromist nebulizer in series at the interface between the developed in-tube PVC SPE device and the ICPMS system to establish a simple, low-contamination, and highly sensitive online microdialysis-in-tube PVC SPE-ICPMS hyphenated system that facilitated the simultaneous in vivo determination of multiple trace metals in rat brain microdialysates. EXPERIMENTAL SECTION Apparatus. The microdialysis-in-tube PVC SPE-ICPMS hyphenated system (Figure 1) consisted of three main parts: the microdialysis probe, the in-tube PVC SPE device, and the ICPMS system. The microdialysis sampling system consisted of a microinjection syringe pump (series 100-53100, Cole Parmer) and a 24mm-long MD probe (CMA/20, CMA, Solna, Sweden) and a 4-mmlong, 0.5-mm-diameter, metal-free polycarbonate (PC) membrane that had a molecular weight cutoff (MWCO) of 20 kDa. The connections of the microinjection syringe pump to the inlet of the microdialysis probe and of the outlet of the microdialysis probe to a micro-Tee (Alltech Associates, Inc., Deerfield, IL) were both accomplished using fluorinated ethylene polypropylene (FEP) tubing (internal volume, 1.2 µL per 100 mm length; length, 10 cm; i.d., 0.12 mm; CMA, Stockholm, Sweden). The dialysate was mixed online with the buffer solution that was perfused by another microinjection syringe pump; the mixture was delivered to an Analytical Chemistry, Vol. 80, No. 18, September 15, 2008
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Table 1. Flow Injection Program Used for the In-Tube PVC SPE-ICPMS System step 1
2
3
4
function fill phosphate-buffered dialysate into PVC tubing
remove waste
detach the analyte and deliver to ICPMS
replace HNO3 with air carrier stream
valve position
medium pumped
flow rate, µL min-1
A: load B: load C: load
480
A: injection B: load C: load
100
mixture of dialysate and buffer
A: injection B: injection C: load
100
HNO3
400
A: injection B: injection C: injection
40
air
400
eight-port valve (C22Z-3186E, Valco, Lucerne, Switzerland) through a 1.5-cm-long, 0.007-in.-i.d. piece of PTFE tubing. The online flow-injection pretreatment system comprised two six-port valves (C2-2348D), and an eight-port valve. All the valves were programmed and controlled by a personal computer via a serial valve interface (SIV-110, Valco). The operational sequence of the online pretreatment system is given in Table 1. The connections and conduits were PTFE connecting tubes. The intube PVC SPE device consisted of a tract of PVC peristaltic tubing (10 cm long × 0.76 mm i.d., Gilson). This PVC pump tube was employed not only to extract the analyte ions but also as a conduit for the dialysate and eluent (0.5% HNO3). The ICP mass spectrometer was an Agilent 7500a system (Agilent, CA). A Micromist nebulizer (AR35-1-EM04EX, Glass Expansion, Victoria, Australia) was fitted to a Scott-type quartz double-pass spray chamber. Because similar results were obtained when analyzing either the peaks’ heights or areas, in this study the samples were quantified only in terms of their peak areas. The instrumental operating conditions selected for optimal sensitivity and low background noise are presented in Table 2. Reagents and Containers. The high-purity water used in this study was purified through deionization and double distillation. The eluent was prepared by dissolving nitric acid (5 mL, ultrapure grade, J.T. Baker, NJ) in high-purity water (1 L). The 40 mM phosphate buffer was prepared by dissolving disodium hydrogen phosphate (5.68 g, Riedel-de Haen, Germany) in high-purity water (1 L) and adjusting the pH using HNO3 solution. The perfusion solution (0.9% NaCl w/v) for microdialysis was prepared by dissolving sodium chloride (0.9 g, ultrapure grade, E. Merck, Darmstadt, Germany) in high-purity water (100 mL). Before each day’s experiments, the phosphate buffer and perfusion solution were purified through a handmade PVC bead column to remove any background contributions. Stock solutions (1000 mg L-1) of analytes were purchased from E. Merck Co. The working aqueous standards were prepared fresh daily using ultrapure normal saline solution. PFA containers were used throughout this study; they were cleaned by immersion overnight in concentrated HNO3 and then overnight in concentrated HCl, followed by steaming successively with HNO3 for 8 h and water vapor for 8 h. The tubes used to connect all of the pieces of apparatus were perfused with high-purity water until the contaminants were eliminated. To avoid contamination of the analyte ions, Norm-Ject plastic perfusion 6962
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dialysate
1.0
buffer
1.0 50
Table 2. Operating Conditions for the Established In-Tube PVC SPE-ICPMS System ICPMS ICPMS spectrometer Ar flow rates outer auxiliary nebulizer makeup plasma forward power sample cone skimmer cone
Agilent 7500a 15 L min-1 0.9 L min-1 1.05 L min-1 0.12 L min-1 1500 W nickel, 1 mm orifice nickel, 0.4 mm orifice
In-Tube Extraction tube material PVC length of tube 10 cm inner diameter of tube 0.76 mm buffer solution 20 mM phosphate buffer, pH 8.0 sample loading 2 µL min-1 removal of waste 50 µL min-1 eluent 0.5% HNO3 elution flow rate 400 µL min-1 Microdialysis Sampling 4 mm × 0.5 mm membrane, metal-free; MWCO ) 20 kDa perfusate 0.9% NaCl solution (pH 7.4) perfusion flow rate 1 µL min-1 sampling time 480 s probe
syringes (Henke Sass Wolf, GmbH; Tufflingen, Germany) were used throughout this study. FTIR Spectroscopy. Polymer-metal complexes were prepared by mixing PVC powder with aqueous solutions containing 2500 mg L-1 of Cu2+ ions, 250 mg L-1 of As3+ ions, and 2500 mg L-1 of Na+ ions, respectively, at pH 8. The mixtures were stirred for 6 h. The resulting solids were filtered, washed with deionized water, and dried at 75 °C. For comparison, pure PVC powder was also treated using the same procedure, but in the absence of the metal ions. After preparation of the polymer-metal complexes, an attenuated total reflectance device (Harrick Scientific Corp., New York) coupled with an FT-IR spectrometer (DA8.3, Bomem) was employed to acquire spectra with the chamber evacuated to sub-millitorr pressures. Several milligrams of PVC or each PVC-metal complex powder was evenly sprinkled on a window of zinc selenite and then scanned from 600 to 1500 cm-1. The characteristic absorption of the C-Cl bonds at 680-780 cm-1 was used to investigate the interactions with the various ions.
Figure 2. FTIR spectra of pure PVC and PVC after reaction with various ions.
In Vivo Experiments. Adult male Sprague-Dawley rats (450-550 g) were obtained from the Laboratory Animal Center of the National Science Council of the Republic of China (Taipei, Taiwan). These animals, which were specifically pathogen-free, were acclimatized to their environmentally controlled quarters (25 °C; 12-h light/12-h dark cycle) for at least 5 days before experimentation and then fasted overnight prior to sacrifice. The rats were fed with a standard diet and water and were treated under the regulations of the “Principles of Laboratory Animal Care” (NIH publication no. 86-23, revised, 1985). The study was approved by the committee of experimental animals of National Tsing-Hua University. To estimate the in vivo concentrations of the analyte elements, it was necessary to determine the recovery of the microdialysis probe, which was accomplished by placing the microdialysis probe in a solution of a known concentration of tested ions and perfusing at 1 µL min-1. The dialysate was online mixed with a stream of phosphate buffer at the same flow rate and then directly introduced to a 10-cm-long PVC tube for the separation and preconcentration; an air carrier stream (flow rate: 50 µL min-1) was used to remove the waste from the PVC tube. Air carrier streams were used to avoid any possible contamination resulting from the use of aqueous carrier streams. After removing the waste, the adsorbed analyte ions from the interior of PVC tube were detached using 0.5% (v/v) HNO3 (ca. 0.66 mL) and transported to the ICPMS system using another air carrier stream (flow rate: 400 µL min-1) for final determination. The probe recovery was calculated by dividing the concentrations in the dialysate by the original concentrations in the tested solution. Linear calibration curves for analyte ions were obtained using external standards from the ion intensities at m/z 55 (Mn), 59 (Co), 60 (Ni), 64 (Zn), 65 (Cu), 114 (Cd) and 208 (Pb). The rats were initially anesthetized with urethane (1200 mg kg-1 of body weight, intraperitoneal injection); they remained anesthetized throughout the experimental period. After mounting
the head of the rat on a stereotaxic apparatus (Davis Kopf Instruments, Tujunga, CA), a midline incision of the skull was executed. The microdialysis probe, which was perfused with saline solution (flow rate: 1.0 µL min-1) was implanted into the exposed brain (5 mm anterior, 5 mm lateral to the bregma, and 5 mm from the brain surface). After 2 h (to reach equilibrium), the basal values of the analytes were monitored continuously, while the concentration was quantified through an in vitro calibration curve that was set up prior to animal testing. Basal Mn, Co, Ni, Cu, Zn, and Pb levels were monitored for at least 200 min prior to administration of MnSO4. After intraperitoneal injection (1000 mg of MnSO4 kg-1 of body weight), the levels of Mn were monitored continuously every 12 min. After finishing the experiment, the probe was removed from the brain and preserved in deionized water. The probe recovery was used only as an index to evaluate the integrity of the membrane; in most cases, it was confined within the range 30-40%. The MD probe was discarded if its probe recovery was less than 20%. RESULTS AND DISCUSSION PVC-Metal Complexation. Functionalized polymers have been used widely for the preconcentration and separation of trace elements from aqueous samples; their analytical application in conjunction with atomic spectrometry is well established.46,47 In this present study, we adopted a nonfunctionalized PVC tube, which we mounted as a peristaltic tube, for initial online separation of trace metal ions from high-salt-content microdialysates collected from the extracellular space of rat brains. To investigate the adsorption behavior of the metal ions of interest toward the interior wall of the PVC tube, we mixed PVC powder individually with Na+, HAsO32-, and Cu2+ ions at pH 8 and then evaporated the solvent prior to FTIR spectroscopic analysis. As indicated in Figure (46) Rao, T. P.; Praveen, R. S.; Daniel, S. Crit. Rev. Anal. Chem. 2004, 34, 177– 193. (47) Wang, J. H.; Hansen, E. H. Trends Anal. Chem. 2005, 24, 1–8.
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Figure 3. Variation of the recovery of analyte ions with respect to pH.
2, the characteristic absorption at 680-780 cm-1, which is associated with skeletal vibration of the C-Cl bonds, shifted to lower frequency only when Cu2+ ions were mixed with the PVC suspension. These results suggest that the interactions between PVC and both Na+ and HAsO32- are negligible, and that the polymer matrix is coordinated to the Cu2+ ions via the chlorine atoms of the polymer. As described in the literature,45,48–51 the dipole-electrostatic interaction between the C-Cl moiety and positive charges on the metal ions is proposed to be the main mechanism to facilitate the complexation retention of transition metal ions when the pH of the sample solution was set to 8. Optimization of Online In-Tube PVC SPE. To optimize the analytical performance of the PVC tubing as a sorbent medium for collecting analyte ions from saline solutions, we investigated the effects of two main parameters on the extraction efficiency: the pH of microdialysate and the sample flow rate. Effect of pH. To determine the basicity of polymers, Watts and Paynter37 used polymers that had been dipped into saline solutions to effect surface ion exchange to evaluate the pKa. In other words, the adsorption of cations onto the surface of a polymer might be affected by the acidity of the sample solution. To investigate the effect of pH on the adsorption of analyte ions on the inner wall of the PVC tubing, normal saline solutions containing analyte ions and buffered with phosphate buffer were transported through the PVC tube at a flow rate of 100 µL min-1. Figure 3 indicates that when the pH of sample solutions was decreased from an initial value of 7, the extraction efficiency of each of the tested elements decreased accordingly. Because the obtained signal intensities of the analyte ions leveled off in the range from pH 7 to 10, for the sake of simplification and maximization of extraction efficiency, (48) Fowkes, F. M.; Tischler, D. O.; Wolfe, J. A.; Lannigan, L. A.; Ademu-John, C. M.; Halliwell, M. J. J. Polym. Sci., Part A: Polym. Chem. 1984, 22, 547– 566. (49) Vrbanac, M. D.; Berg, J. C. J. Adhes. Sci. Technol. 1990, 4, 255–266. (50) Eboatu, A. N.; Alhaji, S. M. J. Therm. Anal. Calorim. 1990, 36, 2383–2391. (51) Paytner, R. W.; Castle, J. E.; Gilding, D. K. Surf. Interface Anal. 1985, 7, 63–68.
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we selected a value of pH of 8 to be appropriate for subsequent experiments. Effect of Sample Flow Rate. Because complete extraction of the analyte ions from the microdialysate was of great importance for improving the analytical performance, we studied the effect of the sample flow rate. In our proposed system, we used air carrier streams to transport the samples; Figure 4 displays the variation in the analyte signals at several different flow rates. We observe that the intensity of the signals obtained decreased upon increasing the flow rate, and that the maximum retention of analyte ions occurred when the sample passed through the PVC tube at flow rates of less than 25 µL min-1. Because the mixed dialysate and buffer solution was transported directly to the PVC tube at a flow rate of 2 µL min-1, quantitative extraction of all of the analyte ions was achieved during the sample loading step. Analytical Characteristics. After optimization of the in-tube PVC SPE procedure, we examined the effects that Na+ ions present in the microdialysate had on the extraction efficiencies of the analyte ions. We found that although the analyte signals obtained were suppressed in the presence of 0.9% (w/v) NaCl, greater than 80% of all of analyte ions could still be extracted from the saline solution, except for Pb2+ ions (72%). Although the salt suppression effect could have been eliminated by decreasing the salinity, we transported the undiluted microdialysate to the PVC tubing for extraction to avoid any possible contamination resulting from the dilution procedure. In addition, we also examined the long-term stability of the in-tube PVC SPE procedure by performing SPE for a continuous 8-h measurement. We found that using the same PVC tube with our proposed online system for measuring analyte ions at 5 µg L-1 resulted in no significant change in the recoveries of the analyte ions. The repeatability of the continuous 8-h measurements for all of the analyte ions was ca. 10% CV, suggesting that this method would be useful in practice. In this study, the loading capacity was determined by flowing a solution containing 100 µg L-1 of Cu2+ ion through a 10-cm PVC tube and measuring the breakthrough volume, revealing a
Figure 4. Signal intensities of analyte ions with respect to the flow rate of sample loading. All the data were normalized to the maximum value.
capacity of 2.34 ng cm-2 (36.5 pmol cm-2). The observed loading capacity was high enough to allow a 10-cm PVC tube to extract about 1100 µg of Cu2+ L-1 from 10 µL of dialysate. The linear dynamic ranges were determined by passing 8 µL portions of standard solutions containing 1 to 10000 µg L-1 of analyte ions. Satisfactory linearities were obtained up to at least 1000 µg L-1, with correlation coefficients higher than 0.995. Traditionally, improving the extraction efficiency and removing any residual matrix remaining in the preconcentration column before and after sample loading requires conditioning and washing of the column, especially for the chelating resin preconcentration procedure. Nevertheless, the need for washing and conditioning steps not only results in a cumbersome and time-consuming procedure but also necessitates the use of large volumes of reagents, which would also tend to cause a high reagent blank. In view of the extremely low basal concentrations of analyte ions in the extracellular fluid of rat brains and the limited sample volume (
