Electrochemically Modulated Liquid−Liquid Extraction of Ionized

Anal. Chem. 2008, 80, 8102–8108

Electrochemically Modulated Liquid-Liquid Extraction of Ionized Drugs under Physiological Conditions Courtney J. Collins, Alfonso Berduque, and Damien W. M. Arrigan* Tyndall National Institute, Lee Maltings, University College, Cork, Ireland Electrochemically modulated liquid-liquid extraction (EMLLE) enables the selective extraction and separation of ions from mixtures by choice of an applied interfacial potential difference. The extraction of ionized drugs from artificial urine is reported in this paper. The artificial urine matrix was characterized by cyclic voltammetry at the interface between two immiscible electrolyte solutions (ITIES), showing that components of that aqueous phase truncate the available potential window at the ITIES. The transfer of three cationic drugs from aqueous artificial urine to the 1,2-dichloroethane organic electrolyte phase was examined. Both propranolol and timolol were found to transfer across the artificial urine-organic interface. However, sotalol transfer was not possible within the available potential window. Extraction of propranolol and timolol from artificial urine into an organogel phase, by electrochemically modulated liquid-liquid extraction, was examined. The application of potentials positive of the drugs’ formal transfer potentials enabled the selective extraction of both propranolol and timolol, with a higher potential being required for timolol. This work demonstrates the practical utility of EMLLE for the selective extraction of target compounds from a complex sample matrix. Liquid-liquid extraction is the one of the most widely used techniques for sample cleanup and analyte preconcentration. The traditional liquid-liquid extraction technique, however, is timeconsuming, needs large volumes of organic solvents, incurs loss of the analyte, and suffers contamination. In recent years, to overcome these drawbacks, attention has been focused on innovations such as liquid-phase microextraction1,2 and microvolume liquid-liquid extraction,3 which offer automation, faster extractions, and reduction in organic solvent consumption. Liquid-liquid extraction techniques can be automated using the flow injection principle. The advantages of this hydrodynamic technique include minimal sample and solvent consumption, enhanced sample throughput, and acceleration of the process.4 * To whom correspondence sould be addressed. E-mail: damien.arrigan @ tyndall.ie. Fax: 353-21-4270271. (1) Pedersen-Bjergaard, S.; Rasmussen, K. E. J. Chromatogr., B 2005, 817, 3–12. (2) Ho, T. S.; Pedersen-Bjergaard, S.; Rasmussen, K. E. Analyst 2002, 127, 608–613. (3) Carlsson, K.; Karlberg, B Anal. Chim. Acta 2000, 423, 137–144. (4) Carlssson, K.; Karlberg, B. Anal. Chim. Acta 2000, 415, 1–7.

8102

Analytical Chemistry, Vol. 80, No. 21, November 1, 2008

A technique frequently applied to flow systems is micro liquidliquid extraction (MLLE), where a droplet of an organic solvent is placed into the aqueous sample and the analytes subsequently partition into the organic droplet via passive diffusion.4 Flow injection analysis offers a wide range of sample pretreatment procedures, which can be carried out online.5 Electrochemistry at the interface between two immiscible electrolyte solutions (ITIES)6-11 has been used for the voltammetric and amperometric determination of ions,12-14 including detection of non-redox-active species.11,14,15 This technique works on the principle of applying an electrical potential difference across the interface formed between two immiscible liquids, each containing dissolved electrolytes. Measurement of the current due to interfacial transfer of ionized species provides the analytical signal. Liquid-liquid electrochemical detection has been employed as the detection method in flow injection analysis and in liquid chromatographic techniques. For example, the chronocoulometric detection of tetraethylammonium ions16 and the amperometric detection of tetrabutylammonium,11 dodecyl sulfate,11 and imipramine17 have been reported. In this paper, focus is centered on the use of liquid-liquid electrochemistry as an extraction method rather than a detection method. Balchen et al.18 recently demonstrated the use of electrokinetic migration across a supported liquid membrane by application of an electrical potential difference. Protonated basic drugs were extracted from samples by application of 300 V dc from an external power supply. However, the present work is concerned only with the application of low applied potentials, with a focus on selective extractions. (5) Motomizu, S.; Korechika, K. Anal. Chim. Acta 1989, 220, 275–280. (6) Samec, Z.; Marecˇek, V.; Weber, J. J. Electroanal. Chem. 1979, 100, 841– 852. (7) Vany´sek, P. Anal. Chem. 1990, 62, 827A–835A. (8) Girault, H. H. Modern Aspects of Electrochemistry; Plenum Press: New York, 1993; Vol. 25, pp 1-62.. (9) Vany´sek, P.; Buck, R. P. J. Electrochem. Soc. 1984, 131 (8), 1792–1796. (10) Vany´sek, P. Trends Anal. Chem. 1993, 12 (9), 357–363. (11) Ortun ˜o, J. A.; Herna´ndez, J.; Sa´nchez-Pedren ˜o, C. Electroanalysis 2004, 16, 827–831. (12) Wilke, S.; Franzke, H; Mu ¨ ller, H. Anal. Chim. Acta 1992, 268, 285–292. (13) Reymond, F.; Fermin, D.; Lee, H. J.; Girault, H. H. Electrochim. Acta 2000, 45, 2647–2662. (14) Lee, H. J.; Pereira, C. M.; Silva, A. F.; Girault, H. H. Anal. Chem. 2000, 72, 5562–5566. (15) Lee, H. J.; Girault, H. H. Anal. Chem. 1998, 70, 4280–4285. (16) Sa´nchez-Pedren ˜o, C.; Ortun ˜o, J. A.; Herna´ndez, J. Anal. Chim. Acta 2002, 459, 11–17. (17) Ortun ˜o, J. A.; Gil, A.; Sa´nchez-Pedren ˜o, C. Sens. Actuators, B: Chem. 2007, 122, 369–374. (18) Balchen, M.; Gjelstad, A.; Rasmussen, K. E.; Pedersen-Bjergaard, S. J. Chromatogr., A 2007, 1152, 220–225. 10.1021/ac800646b CCC: $40.75  2008 American Chemical Society Published on Web 10/09/2008

The main limitation of the ITIES for practical analytical purposes is the mechanical instability of the liquid-liquid interface, especially when employed in flowing solution analysis. Solutions to the problem include jellificiation of the organic phase15,19,20 with poly(vinyl chloride) (PVC), use of dialysis membranes,16,21 plasticized polymeric membranes,11,17 and use of supported liquid membranes.1,18,22-24 Recently,20,25 we reported the use of electrochemistry at the ITIES to control the selective extraction of ions from solutions. The cell employed for this consisted of a stationary organogel phase, over which the mobile aqueous phase flows. The extraction of specific ions from the aqueous phase can be manipulated by control of the applied interfacial potential difference. Each ion has a characteristic transfer potential, based on its Gibbs energy of transfer. Hence, application of a potential difference specific to an ion in a mixture imparts selectivity to the extraction process. This method is referred to as electrochemically modulated liquid-liquid extraction (EMLLE). Although drug detection17,19,26,27 and characterization28,29 has been demonstrated at the liquid-liquid interface using various electrochemical techniques, this paper turns the power of electrochemistry at the ITIES toward the extraction of ionic drugs from a complex matrix that mimics a physiological fluid. The aim was to investigate whether it was possible for positively charged drugs (with differing log P values) to transfer across the liquid-organogel interface and whether these drugs could be selectively extracted from an artificial urine into an organogel phase. The electroextractive transfer behavior of three β-blockers from an artificial urine matrix was examined. The influence of the components of the artificial urine matrix on the electrochemistry at aqueous-organogel interfaces was also assessed. EXPERIMENTAL SECTION Reagents. For artificial urine characterization, the aqueous-phase electrolyte was lithium chloride (10 mM). The organic phase was 1,2-dichloroethane (DCE) containing bis(triphenylphosphoranylidine)ammonium tetrakis(4-chlorophenylborate) (BTPPATPBCl; 10 mM); this salt was prepared by metathesis15,20,30 of bis(triphenylphosphoranylidene) ammonium chloride and potassium tetrakis(4-chlorophenylborate). For EMLLE, the mobile aqueous phase was lithium chloride (10 mM). The organic phase was DCE (19) Fantini, S.; Clohessy, J.; Gorgy, K.; Fusalba, F.; Johans, C.; Kontturi, K.; Cunnane, V. J. Eur. J. Pharm. Sci. 2003, 18, 251–257. (20) Berduque, A.; Sherburn, A.; Ghita, M.; Dryfe, R. A. W.; Arrigan, D. W. M. Anal. Chem. 2005, 77, 7310–7318. (21) Sawada, S.; Torii, H.; Osaki, T.; Kimoto, T. Anal. Chem. 1998, 70, 4286– 4290. (22) Ulmeanu, S. M.; Jensen, H.; Samec, Z.; Bouchard, G.; Carrupt, P. A.; Girault, H. H. J. Electroanal. Chem. 2002, 530, 10–15. (23) Halvorsen, T. G.; Pedersen-Bjergaard, S.; Rasmussen, K. E. J. Chromatogr., A 2001, 909, 87–93. (24) Bjørhovde, A.; Halvorsen, T. G.; Rasmussen, K. E.; Pedersen-Bjergaard, S. Anal. Chim. Acta 2003, 491, 155–161. (25) Berduque, A.; Arrigan, D. W. M. Anal. Chem. 2006, 78, 2717–2725. (26) Kusu, F.; Arai, K. Anal. Sci. 2001, 17, i219-i221. (27) Gulaboski, R.; Cordeiro, M. N. D. S.; Milhazes, N.; Garrido, J.; Borges, F.; Jorge, M.; Pereira, C. M.; Bogeski, I.; Morales, A. H.; Naumoski, B.; Silva, A. F. Anal. Biochem. 2007, 361, 236–243. (28) Reymond, F.; Chopineaux-Courtois, V.; Steyaert, G.; Bouchard, G.; Carrupt, P-A.; Testa, B.; Girault, H. H. J. Electroanal. Chem. 1999, 462, 235–250. (29) Caron, G.; Steyaert, G.; Pagliara, A.; Reymond, F.; Crivori, P.; Gaillard, P.; Carrupt, P-A.; Avdeef, A.; Comer, J.; Box, K. J.; Girault, H. H.; Testa, B. Helv. Chim. Acta 1999, 82, 1211–1222.

containing BTPPATPBCl (10 mM), stabilized by jellification with low molecular weight PVC. The artificial urine31 was composed of calcium chloride dihydrate (1.103 g L-1), sodium chloride (2.295 g L-1), sodium sulfate (2.25 g L-1), potassium phosphate (1.40 g L-1), potassium chloride (1.60 g L-1), ammonium chloride (1.00 g L-1), urea (25 g L-1), and creatinine (1.10 g L-1). All aqueous solutions were prepared in purified water (18 MΩ cm resistivity). The ionic analyte species studied were tetraethylammonium (TEA+) and the β-blocker drugs propranolol hydrochloride, the maleate salt of timolol ((S)-(-)-1-(tert-butylamino)-3-[(4-morpholino-1,2,5-thiadazol-3-yl)oxy]2propranolol), and (±)-sotalol hydrochloride. All samples for injection into the EMLLE system were prepared in artificial urine. All reagents were purchased from Sigma-Aldrich and used as received. Apparatus. All electrochemical investigations were performed using a CHI660B electrochemical analyzer (CH Instruments). For artificial urine characterization, the electrochemical cell32 used was a four-electrode liquid-liquid cell. The interfacial potential difference was applied between a pair of Ag|AgCl reference electrodes. The current was measured by two platinum-mesh counter electrodes (one in each phase). The geometric area of the interface was 1.10 cm2. EMLLE experiments were performed with a four-electrode electrochemical flow cell.24 The interfacial potential difference was applied between a Ag|AgCl reference electrode in the aqueous phase, and a Ag|AgCl pseudo reference electrode in the jellified organic phase. The current was measured by two platinum-mesh counter electrodes (one in each phase). The geometric area of the interface was 1.13 cm2. The flow cell was machined from poly(tetrafluoroethylene) (PTFE) and was held together using PTFE nuts and bolts. The aqueous phase was flowed over the jellified organic phase using a syringe pump (KD Scientific KDS200 series syringe pump). A six-port valve (with a 50-µL injection loop) was used to inject the sample into the flowing aqueous-phase LiCl (10 mM). All potentials reported in this paper are relative to the experimentally used reference electrodes. They were not transformed to the Galvani potential scale. However, for reference, selected voltammograms of TEA+ are shown for which appropriate adjustment to the Galvani scale can be made, based on its position on that scale of +0.44 V in a water/1,2-dichloroethane system.33 RESULTS AND DISCUSSION Artificial Urine Characterization. Initial experiments to characterize the influence of an artificial urine matrix on cyclic voltammetry (CV) at the ITIES showed that the components present in this aqueous mixture decreased the available potential window relative to that achieved when the aqueous phase consisted of lithium chloride (10 mM). The transfer of ions present in the artificial urine matrix significantly reduced the potential window, therefore limiting the working range. In order to fully characterize this truncation of the available potential window, a systematic study of the influence of each component of the artificial urine mixture on the electrochemical (30) Ulmeanu, S.; Lee, H. J.; Fermin, D. J.; Girault, H. H.; Shao, Y. Electrochem. Commun. 2001, 3, 219–223. (31) Griffith, D. P.; Musher, D. M.; Itin, C. Invest. Urol. 1976, 12, 346–350. (32) O’Mahony, A. M.; Scanlon, M. D.; Berduque, A.; Beni, V.; Arrigan, D. W. M.; Faggi, E.; Bencini, A. Electrochem. Commun. 2005, 7, 976–982. (33) Samec, Z. Pure Appl. Chem. 2004, 76, 2147.

Analytical Chemistry, Vol. 80, No. 21, November 1, 2008

8103

Figure 1. Electrochemistry of the individual components of artificial urine at the ITIES. CVs of the blank aqueous-phase electrolyte LiCl (10 mM) (black line), the aqueous phase spiked with the artificial urine components (dark gray line), and the aqueous phase spiked with the artificial urine components and TEA+ (0.048 mM) (light gray line). Artificial urine components and their concentrations: (A) 10 mM KH2PO4; (B) 18.69 mM NH4Cl; (C) 9.7 mM creatinine; (D) 416 mM urea; (E) 3.55 mM CaCl2 · 2H2O; (F) 15.9 mM Na2SO4; (G) 1.07 mM KCl; (H) 50 mM NaCl. Scan rate in all cases was 5 mV s-1.

response at the ITIES was undertaken. The liquid-liquid electrochemical cell contained as its aqueous phase LiCl (10 mM), which was spiked with the individual artificial urine components at their physiological concentrations. TEA+ was also added to each aqueous test phase as a model ion and a potential axis reference ion. The results of this survey are summarized in Figure 1. It can be readily seen that the additions of KH2PO4 (Figure 1A), NH4Cl (Figure 1B), creatinine (Figure 1C) Na2SO4, (Figure 1F), KCl (Figure 1G), and NaCl (Figure 1H) to the aqueous phase all resulted in a shortening of the available potential window at the ITIES. In contrast, the additions of urea and CaCl2 · 2H2O (Figure 1D and E, respectively) did not alter the potential window. The species present in the urine that contribute to shortening of the potential window are known to 8104

Analytical Chemistry, Vol. 80, No. 21, November 1, 2008

transfer across the ITIES, namely, K+,34-36 NH4+,34,35 Na+,34-36 and creatinine. In particular, creatinine addition led to the emergence of a CV response (Figure 1C), which could be used for the detection of creatinine. In comparison to all the other artificial urine components, creatinine transferred at the most negative potential. For the two components not influencing the potential window, both have extremely positive transfer potentials34,36,37 and could therefore be eliminated as the main components transferring in artificial urine. Based on the above observations, creatinine was established as the main contributor to ion-transfer behavior of artificial urine (34) (35) (36) (37)

Osakai, T.; Ogata, A.; Ebina, K. J. Phys. Chem. B 1997, 101, 8341–8348. Katano, H.; Tatsumi, H.; Senda, M. Talanta 2004, 63, 185–193. Osakai, T.; Ebina, K. J. Phys. Chem. B 1998, 102, 5691–5698. Osborne, M. D.; Girault, H. H. Electroanalysis 1995, 7 (8), 714–721.

at the ITIES, due to its least positive transfer potential, but NH4+, K+, and Na+ also contribute to the shortening of the available potential window at the artificial urine | 1,2-dichloroethane interface. Creatinine is the least hydrophilic ion, with the hydrophilicity of NH4+, K+, and Na+ ions increasing in that order. Urea and CaCl2 · 2H2O were seen to not decrease the available potential window. Electrochemistry of β-Blocker Drugs at the Artificial Urine-Organic Interface. The three drug substances chosen for this study, propranolol (pKa 9.23),19 timolol (pKa 9.21)19 and sotalol (pKa1 8.15 and pKa2 9.65),19 are all +1 charged molecules at the pH of the artificial urine, pH 5-6. The transfers of these compounds at the ITIES has been previously reported,19 but not their behavior at the artificial urine - organic electrolyte interface. (a) Electrochemistry of Propranolol. The electrochemical behavior of protonated propranolol was studied using artificial urine as the aqueous phase, at a pH of 5.45. The addition of positively charged propranolol to the artificial urine aqueous phase led to a CV peak at positive potentials (∼+0.5 V, forward sweep), due to propranolol transfer from the aqueous phase into the organic phase. The negative peak on the reverse CV sweep represented the back-transfer of propranolol into the aqueous phase (Figure 2A). The calibration curve (Figure 2A inset) for increasing propranolol concentration showed the peak current response increased linearly in the concentration range studied, 0.025-0.195 mM. The electrochemistry of propranolol (0.23 mM) at increasing scan rates was examined (results not shown). The linear relationship between the peak current and the square root of the scan rate is characteristic behavior of a linear diffusion-controlled electrochemical process, as described by the Randles-Sevcik38 equation (1): ip ) (2.69 × 105)zi3⁄2AD1⁄2Cv1⁄2

(1)

where zi is the net charge of the species, A the interface area (cm2), D the diffusion coefficient of the species studied, in the aqueous phase in this case (cm2 s-1), C the concentration of the species (mol cm-3), and v the voltammetric scan rate (V s-1). The diffusion coefficient of propranolol in artificial urine, using eq 1 and a charge value zi of +1, was found to be 1.0 × 10-5 cm2 s-1, in agreement with the value reported by Fantini et al.,19 (4 ± 1) × 10-6 cm2 s-1. TEA+ was added to the aqueous phase as a reference ion (see Figure 2B) at the end of the experiment. The purpose of TEA+ addition was proof of the appropriate setup of the experimental cell. It can be seen that the transfers of TEA+ and propranolol are overlapped, but that the TEA+ forward and reverse transfers, as indicated by the arrows in Figure 2B, are separated by ∼60 mV, consistent with reversible transfer of a singly charged species. (b) Electrochemistry of Timolol. The electrochemical behavior of timolol in artificial urine was studied at a pH of 5.45. The addition of timolol to the artificial urine aqueous phase produced a CV peak at positive potentials (forward sweep), due to timolol transfer from the aqueous phase into the organic phase. The negative peak in the reverse sweep represented the back(38) Bard, A. J.; Faulkner, L. R. Electrochemical Methods: Fundamentals and Applications, 2nd ed.; Wiley: New York, 2001.

transfer of timolol into the aqueous phase (Figure 2C). The peaks for timolol were not as well-defined as those for propranolol, as timolol transfer occurs closer to the available upper potential limit and the peaks have a resultant greater contribution from background processes (charging current, electrolyte transfers) at the higher potentials. The calibration curve (Figure 2C inset) shows the linear relationship between peak current and concentration over the range studied, 0.025-0.52 mM. The effect of increasing scan rates on the electrochemistry of timolol was investigated (results not shown). The dependence of peak current on the square root of the scan rate demonstrated the linear diffusion-controlled nature of the ion-transfer process. The Randles-Sevcik equation (1) was used to calculate the diffusion coefficient of timolol, assuming an ionic charge of +1. The diffusion coefficient was found to be 1.4 × 10-5 cm2 s-1, in agreement with the value of Fantini et al.19 of (5 ± 1) × 10-6 cm2 s-1. TEA+ was again added to the aqueous phase as a reference ion (see Figure 2D) at the end of the experiment to verify adequate experimental setup. Two sets (forward and reverse) of peaks were observed, one set for each ion. TEA+ transfer occurred at less positive potentials (+0.43 V), in comparison to timolol transfer at more positive potentials (+0.58 V). TEA+ is less hydrophilic than timolol, explaining its transfer at less positive potentials; this also confirms that timolol is more hydrophilic than propranolol, as the latter’s transfer peaks were severely overlapped by those of TEA+ (Figure 2B). The transfer of TEA+ (Figure 2D) exhibited a ∼60 mV peak-peak separation, as expected for a simple, reversible iontransfer process with one positive charge. (c) Electrochemistry of Sotalol. On examination of the electrochemical behavior of sotalol in artificial urine at a pH of 5.45, it was found that there were no transfer peaks for sotalol (see Figure 2E). However, at the positive end of the potential window, a significant increase in current was observed. Sotalol transfer may thus be occurring at the extremity of the available potential window at the artificial urine-DCE interface and overlapping with the transfers of the urine components such as creatinine, K+, and NH4+ ions. Compared to propranolol and timolol, sotalol is the least lipophilic drug. More positive applied potentials are therefore needed for sotalol to transfer, and under the present conditions, directly in artificial urine, it is not possible to detect it. The trend in transfer potentials in the above experiments was found to be propranolol < timolol < sotalol at the artificial urine | 1,2-dichloroethane interface, which was in agreement with the trend for the three drugs reported by Fantini et al.19 at a gelled aqueous | 1,2-dichloroethane interface. The trend depends on the lipophilicity of the drugs, with the least hydrophilic drug transferring across the interface at the lowest potential. The lipophilicity of propranolol and timolol at a water | DCE interface has been calculated previously by Caron et al.29 for both their neutral and cationic forms. For both forms, it was found that propranolol was the more lipophilic of the two drugs. As discussed previously,19 propranolol is the most lipophilic due to its naphthalene group, timolol being the next lipophilic due to both hydrophilic and lipophilic substituents, and sotalol being the least lipophilic due to its ability to hydrogen bond with water via its sulfonamido moiety. The observed electrochemical behavior in this work is in Analytical Chemistry, Vol. 80, No. 21, November 1, 2008

8105

Figure 3. Potentiostatic extraction of propranolol (1 mM) from artificial urine: (A) E ) 0.0 V, (B) E ) 0.35 V, (C) E ) 0.55 V. Triplicate extractions shown. The flowing phase was aqueous 10 mM LiCl, into which the sample prepared in artificial urine was injected.

Figure 2. Electrochemistry of drugs at the artificial urine 1,2dichloroethane interface. (A) Propranolol in artificial urine (pH 5.45): (1) 0, (2) 0.025, (3) 0.05, (4) 0.075, (5) 0.12, and (6) 0.195 mM. Inset: calibration curve of forward peak current versus concentration. (B) Propranolol and TEA+ in artificial urine (pH 5.45): blank (black) and 0.23 mM propranolol with 0.23 mM TEA+ (light gray). (C) Timolol in artificial urine (pH 5.45): (1) 0, (2) 0.025, (3) 0.05, (4) 0.1, (5) 0.15, (6) 0.24, (7) 0.34, (8) 0.43, and (9) 0.52 mM. Inset: calibration curve of forward peak current versus timolol concentration. (D) Electrochemistry of timolol and TEA+ in artificial urine (pH 5.45): blank (black) and 0.1 mM timolol with 0.05 mM TEA+ (light gray). Scan rate: 5 mV s-1. (E) Sotalol in artificial urine (pH 5.45): blank (black), 0.7 mM sotalol (dark gray), and sotalol with TEA+ (thin light gray line). Scan rate in all cases was 5 mV s-1. 8106

Analytical Chemistry, Vol. 80, No. 21, November 1, 2008

agreement with the trends in the lipophilic nature of the compounds. Electrochemically Modulated Liquid-Liquid Extraction. The ion-transfer potentials of propranolol and timolol between artificial urine and DCE are now known from the CV studies described above, so by applying specific potentials, these drugs can be selectively extracted from artificial urine using the recently described method of EMLLE.20,25 Application of a potential positive of the formal ion-transfer potential for the cationic drugs causes the drugs to transfer into the organogel phase. An applied potential negative of the formal transfer potential for the cationic drugs causes the cationic drugs to remain in the aqueous phase or to be back extracted into the aqueous phase from the organogel phase (if previously extracted). (a) Potentiostatic Extraction of Propranolol from Artificial Urine. At the lowest applied potential employed, 0.0 V (see Figure 3A), low-magnitude negative peak currents were observed, for injections of both artificial urine and propranolol in artificial urine. The artificial urine was injected as the control experiment. The presence of negative peak currents of similar magnitude indicated that no extraction was occurring, because the potential applied was less than that required for ionized drug transfer. The extraction of propranolol did occur at more positive potentials of

Figure 4. Postextraction analysis by CV: blank of aqueous phase (i.e., LiCl (10 mM) and BTPPATPBCl in DCE-PVC organogel phase (black line) and extracted propranolol (gray line) following extraction of propranolol (1 mM) from artificial urine into the organogel phase. CVs were performed under stationary conditions. Scan rate: 5 mV s-1.

E ) 0.35 and 0.55 V (see Figure 3B and C, respectively), producing large positive peak currents, in comparison to the much smaller artificial urine peak currents. A leveling-off of the propranolol extraction currents was observed at applied potentials of E ) 0.50 and 0.55 V as the peak current magnitudes for both were quite similar. Maximum propranolol extraction was achieved at these applied potentials. Postextraction CV analysis, under stationary conditions, (i.e., after the potentiostatic extraction of propranolol from artificial urine at an applied potential of E ) 0.35 V), verified that propranolol was extracted into the organogel phase (see Figure 4), with the forward peak maximum at ∼0.3 V. This forward (negative current) peak was due to the transfer of the propranolol into the aqueous phase from the organogel phase, and the positive peak (positive sweep) was due to its re-extraction into the organogel phase. For these postextraction CV analyses, the analyte in question is not present in the aqueous phase but in the organogel phase. Thus, the response measured is due to its iontransfer behavior, with the transfer processes from organogel to aqueous phase, and the reverse, being what control the observed CV. (b) Potentiostatic Extraction of Timolol from Artificial Urine. When an applied potential of E ) 0.0 V was used, no extraction of timolol was achieved, as this potential was below that required to effect transfer of the ionized drug across the ITIES. However, significant negative peak currents were obtained, possibly due to transfer of the maleate anion associated with this drug in its commercially available form. The extraction current increased when the applied interfacial potential difference was made more positive, as a result of timolol extraction. The positively charged timolol ion was induced to transfer at more positive applied potentials, providing a difference in peak current magnitudes between artificial urine and the mixture of timolol in artificial urine. An increase in positive applied potentials (e.g., E ) 0.55 V) resulted in an increase in timolol extraction (see Figure 5). Applied potentials of E ) 0 and 0.05 V did not result in timolol extraction (not shown). CV analysis of timolol after its potentiostatic extraction into the organogel phase at an applied potential of E ) 0.4 V produced a voltammetric response (with forward and reverse peaks) due to ion-transfer voltammetry of the previously extracted timolol.

Figure 5. Potentiostatic extraction of timolol (1 mM) from artificial urine, E ) 0.55 V. Other conditions as for Figure 3.

(c) Potentiostatic Extraction of TEA+ from Artificial Urine as a Control Experiment. Control experiments on injections of artificial urine into the flow cell resulted in initial negative current transients, followed by a positive current transient at an applied potential of E ) 0.05 V. The magnitudes of these current peaks were extremely low and reflect background processes occurring rather than transfer of specific ions. The injection of a mixture of TEA+ (1 mM) in artificial urine into the flow cell was carried out as a model experiment. This produced amperometric peaks of very low magnitude when low potentials were applied. At higher potentials, the positive peak current for both artificial urine and the mixture of TEA+ (1 mM) in artificial urine increased in magnitude. At applied potentials of E ) 0.30 V and E ) 0.35 V, the peak currents for the mixture of TEA+ in artificial urine were much larger than those of the artificial urine solution (not shown). This was due to the fact that the TEA+ ion in the artificial urine matrix was extracted into the organogel phase, whereas in the case of artificial urine only, background processes were occurring. Postextraction CV analysis under stationary solution conditions demonstrated the detection of TEA+ previously extracted into the organogel phase (not shown), with a midpoint potential of ∼0.2 V. These CV analyses postextraction usefully verify the presence of the extracted ion in the organogel phase, as the CV is carried out without that ion in the aqueous phase, so that the response recorded is due to the presence of the extracted ion in the organic phase.20,25 These results clearly demonstrate that a model ion used widely in electrochemical studies of the ITIES can be extracted from a realistic matrix such as artificial urine using EMLLE. (d) Hydrodynamic Voltammograms. The extraction capability of diverse analytes at a given applied potential can be compared using hydrodynamic voltammograms (HDVs). Figure 6 compares the extraction capabilities of the two drug cations from the artificial urine matrix studied. Propranolol extraction occurred for E > 0.3 V, but at lower potentials it was not extracted. Timolol displayed different extraction capabilities. At less positive potentials (i.e., E ) 0.0 V), the maleate component (negatively charged) of the timolol salt used was observed to transfer. However, at potentials positive of 0.35 V, cationic timolol itself was extracted. Thus HDVs provide a convenient means of comparison of the extractive properties of different analytes and enable selection of suitable potentials for selective or nonselective extractions to be effected. By consideration of the total charge under the current transients Analytical Chemistry, Vol. 80, No. 21, November 1, 2008

8107

(corrected for any instability in the reference electrode systems by referral to the reversible transfer potential for TEA+) were in the region of 0.05 V. It was also found that the lower pH matrix resulted in a further shortening of the available potential window, due to transfer of protons at the positive limit.

Figure 6. Hydrodynamic voltammograms for extraction of propranolol (black line) and timolol (gray line) from artificial urine into the organogel phase. Other conditions as stated for Figure 3.

used to construct Figure 6, and comparison to the mole amounts injected (by use of Faraday’s law), the extraction efficiencies for this cell were found to be 10% of the injected amount. Alternate cell designs will be investigated in order to achieve improved extraction efficiency. (e) Effects of Varying pH and Ionic Strengths of Artificial Urine. A preliminary investigation of the effects of pH and ionic strength variations of artificial urine on the extraction of propranolol was undertaken. It has been reported39 that the pH of urine may vary between pH 4 and 8, depending on physiological condition. Similarly, the ionic strength of the urine may vary between 10 and 800 mM.40,41 In this work, it was found that lower pH (e.g., pH 3.7) and higher ionic strength (e.g., addition of 500 mM NaCl to the artificial urine matrix) shifted the transfer potential of propranolol to lower potentials, while higher pH (e.g., pH 8.0) and lower ionic strength (e.g., addition of 5 mM NaCl to the artificial urine matrix) shifted the transfer potential to higher potentials. However, the magnitude of these potential shifts (39) Souhami, R. L., Moxham, J., Eds. Textbook of Medicine, 2nd ed.; Churchill Livingstone: Edinburgh, 1994. (40) Ronteltap, M.; Maurer, M.; Gujer, W. Water Res. 2007, 41, 977–984. (41) Raichur, A.; Watkinspitchford, M. Proc. Ann. Int. Conf. IEEE Eng. Med. Biol. Soc. 1992, 14, 2718–2719.

8108

Analytical Chemistry, Vol. 80, No. 21, November 1, 2008

CONCLUSIONS The studies reported demonstrate that electrochemistry at the ITIES can be applied for the extraction of ions from complex matrices. In this case, a model ion, TEA+, and specific drug molecules were extracted from a multicomponent mixture, artificial urine. Although the use of artificial urine as the aqueous phase in an ITIES electrochemical cell decreased the available potential window for studies of ion-transfer process, species whose transfer potential falls within the window can be extracted. Protonated drug molecules, specifically propranolol and timolol, can be extracted from artificial urine by appropriate choice of applied interfacial potential differences. It has been found that application of potentials positive of the formal transfer potential of the drugs causes their extraction into the organogel phase. This leads to the possibility of this electrochemical technique being used for cleanup and preparation of complex samples for instrumental analysis. Such a strategy can lead to more easily controlled extraction processes and to their interfacing with a range of instrumental detection methods. However, an issue that needs to be improved is the extraction efficiency. Improved cell designs and three-dimensional ITIES may provide such an improvement. ACKNOWLEDGMENT This work was carried out with the financial support of Science Foundation Ireland (grants 02/IN.1/B84 and 07/IN.1/B967).

Received for review March 31, 2008. Accepted August 18, 2008. AC800646B