Ion-Transfer Voltammetric Determination of the β-Blocker Propranolol

Feb 12, 2009 - ployed a preconditioning step as well as a preconcentra- tion step followed by analytical signal generation based on the back-transfer ...
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Anal. Chem. 2009, 81, 2344–2349

Ion-Transfer Voltammetric Determination of the β-Blocker Propranolol in a Physiological Matrix at Silicon Membrane-Based Liquid|Liquid Microinterface Arrays Courtney J. Collins and Damien W. M. Arrigan* Tyndall National Institute, Lee Maltings, University College, Cork, Ireland In this work, the ion-transfer voltammetric detection of the protonated β-blocker propranolol in artificial saliva is presented. Cyclic voltammetry, differential pulse voltammetry, and differential pulse stripping voltammetry (DPSV) were employed in the detection of the cationic drug based on ion-transfer voltammetry across arrays of microinterfaces between artificial saliva and an organogel phase. It was found that the artificial saliva matrix decreased the available potential window for ion-transfer voltammetry at this liquid|liquid interface but transfer of protonated propranolol was still achieved. The DPSV method employed a preconditioning step as well as a preconcentration step followed by analytical signal generation based on the back-transfer of the drug across the array of microinterfaces. The DPSV peak current response was linear with drug concentration in the artificial saliva matrix over the concentration range of 0.05-1 µM (ip ) -8.13 (nA µM-1)(concentration) + 0.07 (nA), R ) 0.9929, n ) 7), and the calculated detection limit (3sb) was 0.02 µM. These results demonstrate that DPSV at arrays of liquid|liquid microinterfaces is a viable analytical approach for pharmaceutical determinations in biomimetic matrixes. Drug detection has traditionally been investigated in matrixes such as blood and urine, but recently saliva has become a popular alternative due to the noninvasive manner in which samples may be collected and the difficulty associated with tampering with the sampling process. Interestingly, saliva contains only ∼0.3% protein (mostly enzymatic), thus minimizing the effect of drug-protein binding that largely occurs in plasma.1 The secretion of drugs into saliva is via passive diffusion2 and depends on the drugs’ pKa, lipophilicity, plasma protein binding property, and of course the pH of saliva.3 It has been reported that saliva can be used as an alternative to blood for drug detection when drug concentration in saliva is almost equal to the unbound drug * To whom correspondence should be addressed. E-mail: damien.arrigan@ tyndall.ie. Fax: 353-21-4270271. (1) Townsend, S.; Fanning, L.; O’Kennedy, R. Anal. Lett. 2008, 41, 925–948. (2) Haeckel, R.; Hanecke, P. Eur. J. Clin. Chem. Clin. Biochem. 1996, 34, 171– 191. (3) Skopp, G.; Potsch, L. Int. J. Legal Med. 1999, 112, 213–221.

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content of plasma.4 Consequently, drugs are usually assigned a saliva/plasma drug concentration ratio (S/P),2 which reflects the concentration ratio of the unbound drug found between saliva and plasma. Propranolol has an S/P ratio of 0.5,5 meaning that the concentration of drug found in saliva is half that of the unbound propranolol in plasma. In recent years illicit drugs and their metabolites6 have been detected in saliva by use of commercially available immunoassay kits. Therapeutic drugs have also been determined in saliva to study the mechanism of drug transfer5 and for detection purposes.7-9 Interest in the use of electrochemistry at the interface between two immiscible electrolyte solutions (ITIES)10-13 has been of growing interest in the last few decades due to the fact that this interface is recognized as a simple model of one side of a biological membrane and proves useful for drug lipophilicity14 and partition coefficient (log P)15 measurements. Therefore, liquid-liquid electrochemistry can play an important role in the areas of pharmaceutical chemistry, medicine, and pharmacology. Although charged drug species have been electrochemically detected at the macro-ITIES,16-19 electrochemical detection of drugs at microITIES array has not been reported. In a similar manner to the improvements in electroanalytical performances achieved by miniaturization of solid electrodes to (4) Wolff, K.; Farrell, M.; Marsden, J.; Monteiro, M. G.; Ali, R.; Welch, S.; Strang, J. Addiction 1999, 94, 1279–1298. (5) Hold, K. M.; Deboer, D.; Soedirman, J. R.; Zuidema, J.; Maes, R. A. A. J. Pharm. Biomed. Anal. 1995, 13, 1401–1407. (6) Jenkins, A. J.; Oyler, J. M.; Cone, E. J. J. Anal. Toxicol. 1995, 19, 359–374. (7) Mahajan, P.; Grech, E. D.; Ridgway, E. J.; Turner, P.; Pearson, R. M. Br. J. Clin. Pharmacol. 1984, 17, P185–P186. (8) Mahajan, P.; Grech, E. D.; Pearson, R. M.; Ridgway, E. J.; Turner, P. Br. J. Clin. Pharmacol. 1984, 18, 849–852. (9) Groschl, M.; Kohler, H.; Topf, H. G.; Rupprecht, T.; Rauh, M. J. Pharm. Biomed. Anal. 2008, 47, 478–486. (10) Samec, Z.; Marecek, V.; Weber, J. J. Electroanal. Chem. 1979, 100, 841– 852. (11) Vanysek, P.; Buck, R. P. J. Electrochem. Soc. 1984, 131, 1792–1796. (12) Ortuno, J. A.; Hernandez, J.; Sanchez-Pedreno, C. N. Electroanalysis 2004, 16, 827–831. (13) Arrigan, D. W. M. Anal. Lett. 2008, 41, 3233–3252. (14) Bouchard, G.; Carrupt, P. A.; Testa, B.; Gobry, V.; Girault, H. H. Chem.sEur. J. 2002, 8, 3478–3484. (15) Bouchard, G.; Galland, A.; Carrupt, P. A.; Gulaboski, R.; Mirceski, V.; Scholz, F.; Girault, H. H. Phys. Chem. Chem. Phys. 2003, 5, 3748–3751. (16) Gulaboski, R.; Cordeiro, M.; 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. (17) Fantini, S.; Clohessy, J.; Gorgy, K.; Fusalba, F.; Johans, C.; Kontturi, K.; Cunnane, V. J. Eur. J. Pharm. Sci. 2003, 18, 251–257. 10.1021/ac802644g CCC: $40.75  2009 American Chemical Society Published on Web 02/12/2009

microelectrodes and microelectrode arrays,20-23 electrochemistry at miniaturized ITIES (often referred to as micro-ITIES) has also been developed.24,25 Microinterface arrays have also been developed, based on laser-ablated holes in thin polymer sheets which were used to define the interface between the two electrolyte phases.26 These microhole interfaces were shown to behave like inlaid microdiscs.27 The advantages of these microinterfaces over macrointerfaces were a reduced ohmic drop, a reduced charging current, enhanced mass transport, and an improved sensitivity.28,29 Liquid-liquid microinterface arrays have been typically fabricated using polymer membranes or films (i.e., poly(ethylene terephthalate), PET),28,30-34 but recently the use of micropore silicon membranes produced by photolithography and silicon etching processes have been developed35 and shown to be useful in the detection of dopamine and oligopeptides by facilitated ion transfer.36-38 Microinterface arrays may also be created by use of oil droplets distributed across a solid electrode surface.39-41 The aim of this present work was to examine the use of electrochemistry at micro-ITIES arrays as an analytical tool for drug detection in a complex matrix that mimics a biological fluid, in this case artificial saliva. This builds on previous work on electrochemically modulated liquid-liquid extraction of drugs from artificial urine matrix.42,43 In this paper, the transfer and (18) Ortuno, J. A.; Sanchez-Pedreno, C.; Gil, A. Anal. Chim. Acta 2005, 554, 172–176. (19) Ortuno, J. A.; Gil, A.; Sanchez-Pedreno, C. Sens. Actuators, B 2007, 122, 369–374. (20) Zoski, C. G. Electroanalysis 2002, 14, 1041–1051. (21) Berduque, A.; Lanyon, Y. H.; Beni, V.; Herzog, G.; Watson, Y. E.; Rodgers, K.; Stam, F.; Alderman, J.; Arrigan, D. W. M. Talanta 2007, 71, 1022– 1030. (22) Ordeig, O.; del Campo, J.; Munoz, F. X.; Banks, C. E.; Compton, R. G. Electroanalysis 2007, 19, 1973–1986. (23) Beni, V.; Arrigan, D. W. M. Curr. Anal. Chem. 2008, 4, 229–241. (24) Taylor, G.; Girault, H. H. J. J. Electroanal. Chem. 1986, 208, 179–183. (25) Shao, Y.; Osborne, M. D.; Girault, H. H. J. Electroanal. Chem. 1991, 318, 101–109. (26) Campbell, J. A.; Girault, H. H. J. Electroanal. Chem. 1989, 266, 465–469. (27) Osborne, M. C.; Shao, Y.; Pereira, C. M.; Girault, H. H. J. Electroanal. Chem. 1994, 364, 155–161. (28) Lee, H. J.; Beattie, P. D.; Seddon, B. J.; Osborne, M. D.; Girault, H. H. J. Electroanal. Chem. 1997, 440, 73–82. (29) Liu, B.; Mirkin, M. V. Electroanalysis 2000, 12, 1433–1446. (30) Wilke, S.; Osborne, M. D.; Girault, H. H. J. Electroanal. Chem. 1997, 436, 53–64. (31) Beriet, C.; Girault, H. H. J. Electroanal. Chem. 1998, 444, 219–229. (32) Osborne, M. D.; Girault, H. H. Mikrochim. Acta 1995, 117, 175–185. (33) Qian, Q. S.; Wilson, G. S.; Bowman-James, K.; Girault, H. H. Anal. Chem. 2001, 73, 497–503. (34) Qian, Q. S.; Wilson, G. S.; Bowman-James, K. Electroanalysis 2004, 16, 1343–1350. (35) Zazpe, R.; Hibert, C.; O’Brien, J.; Lanyon, Y. H.; Arrigan, D. W. M. Lab Chip 2007, 7, 1732–1737. (36) Berduque, A.; Zazpe, R.; Arrigan, D. W. M. Anal. Chim. Acta 2008, 611, 156–162. (37) Scanlon, M. D.; Herzog, G.; Arrigan, D. W. M. Anal. Chem. 2008, 80, 5743– 5749. (38) Strutwolf, J.; Scanlon, M. D.; Arrigan, D. W. M. Analyst 2009, 134, 148– 158. (39) Rayner, D.; Fietkau, N.; Streeter, I.; Marken, F.; Buckley, B. R.; Page, P. C. B.; del Campo, J.; Mas, R.; Munoz, F. X.; Compton, R. G. J. Phys. Chem. C 2007, 111, 9992–10002. (40) Barnes, A. S.; Fietkau, N.; Chevallier, F. G.; del Campo, J.; Mas, R.; Munoz, F. X.; Jones, T. G. J.; Compton, R. G. J. Electroanal. Chem. 2007, 602, 1–7. (41) MacDonald, S. M.; Fletcher, P. D. I.; Cui, Z. G.; Opallo, M. C.; Chen, J. Y.; Marken, F. Electrochim. Acta 2007, 53, 1175–1181. (42) Collins, C. J.; Berduque, A.; Arrigan, D. W. M. Anal. Chem. 2008, 80, 8102– 8108.

detection of a cationic β-blocker drug, protonated propranolol, is reported by employing cyclic voltammetry (CV), differential pulse voltammetry (DPV), and differential pulse stripping voltammetry (DPSV) as the detection methods. Analytical method development included an analysis of the effect of a preconditioning step on voltammetric behavior, so as to obtain extensive cleaning of the organogel phase between experiments, and the effect of preconcentration times on the detection limit. Nanomolar detection in a biomimetic fluid has been achieved. EXPERIMENTAL SECTION Reagents. All the reagents were purchased from SigmaAldrich and used as received except for the following: magnesium pyrophosphate (Alfa Aesar), urea (Qiagen), and 1,6-dichlorohexane (1,6-DCH) (Merck). The latter was purified according to the published procedure.44 For the macro-ITIES, the aqueous phase electrolyte was lithium chloride (10 mM). The organic phase was 1,2-dichloroethane (DCE) containing bis(triphenylphosphoranylidine)ammonium tetrakis(4-chlorophenylborate) (BTPPATCB, 10 mM); this salt was prepared by metathesis28,45 of bis(triphenylphosphoranylidene)ammonium chloride (BTPPACl, Aldrich) and potassium tetrakis(4-chlorophenyl-borate) (KTCB, Fluka). For studies of drug detection at the micro-ITIES array, the aqueous phase was artificial saliva,46,47 composed of magnesium pyrophosphate (0.0016 g L-1), carboxymethyl cellulose (4 g L-1), urea (4 g L-1), disodium hydrogen phosphate (0.6 g L-1), anhydrous calcium chloride (0.6 g L-1), potassium chloride (0.4 g L-1), and sodium chloride (0.4 g L-1). The organogel phase was prepared using BTPPATCB in 1,6-DCH and low molecular weight poly(vinyl chloride) (PVC).37,48 All aqueous solutions were prepared in purified water (resistivity of 18 MΩ cm). The β-blocker propranolol hydrochloride was purchased from SigmaAldrich and used as received. Apparatus. All experiments were performed with a CHI660B electrochemical analyzer (CH Instruments, Texas). Characterization of the effect of the artificial saliva on electrochemistry at the ITIES was performed at both macro-ITIES and micro-ITIES array. For the former, the electrochemical cell49 used was a fourelectrode liquid-liquid cell. The interfacial potential difference was applied between a pair of Ag|AgCl reference electrodes. The current was measured by two platinum counter electrodes (one in each phase). The geometric area of the interface was 1.10 cm2. For the micro-ITIES array experiments, the micropore arrays37 were fabricated in silicon using photolithographic patterning and a combination of wet and dry silicon etching, as described previously.35 The fabrication procedure provided micropores with hydrophobic walls so that the organic phase filled the pores.35,38 The micropore array consisted of eight micropores arranged in a hexagonal close-packed arrangement, each with a diameter of 52 µm and a pore center-to-center distance of 500 µm. The mi(43) Collins, C. J.; Arrigan, D. W. M. Anal. Bioanal. Chem. 2009, 393, 835845. (44) Katano, H.; Tatsumi, H.; Senda, M. Talanta 2004, 63, 185–193. (45) Lee, H. J.; Girault, H. H. Anal. Chem. 1998, 70, 4280–4285. (46) Fusayama, T.; Nomoto, S.; Katayori, T. J. Dent. Res. 1963, 42, 1183 ff. (47) Vahed, A.; Lachman, N.; Knutsen, R. D. Dent. Mater. 2007, 23, 855–861. (48) Osakai, T.; Kakutani, T.; Senda, M. J. Electrochem. Soc. 1987, 134, C520– C520. (49) 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.

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croporous silicon membranes were sealed onto the lower orifice of glass cylinders using a silicone rubber (RS 555-588 silicon rubber compound). The gellified organic phase solution was introduced into the silicon micropores via the glass cylinder, and then the organic reference solution was placed on top of the gellified organic phase. The silicon membrane was then immersed into the aqueous phase (artificial saliva), and voltammetry experiments were carried out. The waveform parameters used for DPV and DPSV were as follows: pulse amplitude, 0.05 V; sampling width, 0.055 s; step height, 4 mV. These were applied for all the differential pulse voltammetry experiments. RESULTS AND DISCUSSION Artificial Saliva Characterization. Initial experiments to characterize the influence of the artificial saliva matrix on CV at the macro-ITIES and at the micro-ITIES array showed that the components present in this aqueous mixture decreased the available potential window relative to that achieved when the aqueous phase consisted solely of LiCl (10 mM). This decrease of the potential window limited the working potential range for drug detection. To fully characterize which artificial saliva components truncated the potential window, the influence of each component on the electrochemical response at the ITIES was studied individually. The macro liquid-liquid electrochemical cell contained LiCl (10 mM) as its aqueous phase which was spiked with the individual components of artificial saliva at their physiological concentrations. It was observed that urea, magnesium pyrophosphate, carboxymethyl cellulose, calcium chloride, and disodium hydrogen phosphate did not alter the potential window. However, potassium chloride and sodium chloride resulted in the shortening of the available potential window (CVs not shown). At the microITIES array, in which the organic electrolyte phase was gellified 1,6-DCH, it was found that only potassium ions caused a shortening of the available potential window. On the basis of these observations, potassium chloride and sodium chloride are the main contributors to the ion-transfer behavior of artificial saliva at the ITIES. Voltammetric Analysis at the Micro-ITIES Array. The diffusion profile for ion transfer across the micro-ITIES has been studied by Shao et al.25 for micropipettes. It was observed that radial diffusion occurred when the ion-transfer process was controlled by the species entering the micropipette and that linear diffusion occurred when the transfer process was controlled by the species leaving the micropipette. These diffusion fields were also observed in the micropore array geometry employed in these experiments35,38 due to the fact that the micropore center-to-center distances are large enough to prevent overlap of the individual diffusion fields at each micropore. CV analysis of 100 µM propranolol in artificial saliva (see Figure 1A) produced an asymmetric CV shape with an apparent steady-state curve on the forward sweep and a peak-shaped response on the reverse sweep. The pH of the artificial saliva (pH ∼ 7) was maintained below the pKa of propranolol (pKa 9.23),17 thus ensuring it was protonated and enabling it to transfer across and be detected at the micro-ITIES array. Spherical diffusion of propranolol was the controlling mass-transport process occurring toward the micropores on the aqueous side of the membrane. The back-transfer of propranolol from the organogel phase (negative sweep) was controlled by linear 2346

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Figure 1. Electrochemistry of 100 µM propranolol in artificial saliva at the micro-ITIES array using (A) cyclic voltammetry, scan rate, 10 mV s-1; blank (artificial saliva) (black), 100 µM propranolol (gray); (B) linear sweep voltammetry, scan rate, 100 mV s-1; blank (artificial saliva) (black), 100 µM propranolol (gray).

diffusion within the micropores, as demonstrated by a peakshaped current. As a result, two different response current shapes were observed for the transfer (sigmoidal) and backtransfer (linear) of propranolol across the micro-ITIES, consistent with the different diffusion fields on either side of the membrane, as discussed previously.25,35,38 The CV behavior indicates that the micropores of the membrane were filled with organogel resulting in an inlaid geometry at the aqueous side of the membrane. The microinterfaces thus displayed mass-transport behavior and current responses similar to inlaid solid microdisc electrodes27 according to Ilim ) n4ziFDCr

(1)

where Ilim is the limiting current, n is the number of pores, zi is the charge of the transferring ion species, F is the Faraday constant, D is the diffusion coefficient, C is the bulk concentration of the transferring species, and r is the radius of the pore (26 µm). The diffusion coefficient for propranolol in artificial saliva was determined, by use of eq 1 and the limiting current from the CV in Figure 1A, to be 1.15 × 10-5 cm2 s-1, in agreement with the figure of Fantini et al.17 of 5 ± 1 × 10-6 cm2 s-1 (in aqueous LiCl) and with the value for diffusion in artificial urine, 1.0 × 10-5 cm2 s-1.42 As can be seen in Figure 1, however, the forward ion-transfer process is not purely controlled by radial diffusion: there is a slight peak in the CV forward sweep while the linear sweep voltammetry shown in Figure 1B shows a more pronounced peak shape. This is attributed to a contribution from linear diffusion due to the size of the micropores, 52 µm diameter, which was also observed in a simulation study.38

Figure 2. Method of investigating the effect of preconditioning times on the cleaning of the organogel phase between experiments.

The CV currents observed for the transfer and back-transfer of propranolol were proportional to the concentration of the analyte transferring. Therefore, by consideration of the total charge under the current transients, the number of moles transferring and backtransferring were determined using Faraday’s law Q ) mziF

(2)

where Q is the total charge, m is the number of moles, and the other parameters as defined above. The number of moles of propranolol transferred from the aqueous artificial saliva into the organogel phase was determined to be 3 × 10-12 mol, whereas the amount back-transferred was only 1.5 × 10-12 mol. This suggests that a substantial proportion of the transferred propranolol remains in the organogel phase within the micropores and is not transferred back to the aqueous artificial saliva phase during the reverse sweep of the CV. Indeed, a recent simulation study of CV at these microITIES arrays has revealed that some of the transferred species remain within the organic phase and does not back-transfer.38 This may be a consequence of the high viscosity of the organogel phase. This observation implies that organogel cleaning or conditioning between repetitive voltammetric analysis measurements will be crucial to prevent the effects of sample carryover and contamination. This effect was therefore studied, as reported below, and referred to as preconditioning time. Influence of the Preconditioning Time on Organogel Cleaning. A preconditioning step may be employed in an analytical procedure prior to the next step in a series of repeated

measurements for the purpose of removing any analyte that is not back-extracted during an electrochemical measurement step. Hence in the present experimental arrangement, preconditioning is the application of a potential more negative than the transfer potential of the analyte being studied for a given amount of time. The applied potential is a potential at which the analyte will be expelled from the organogel phase thereby cleaning the gel. Initially, the effect of a preconditioning period on the voltammetric response was examined by using a 15 s preconditioning time. It was found in stripping voltammetry experiments subsequent to the preconditioning period that the peak current did not increase with the concentration of the analyte. This was attributed to the buildup of the analyte concentration in the organogel after multiple experiments (all concentration levels were analyzed in quadruplicate). As a result, the influence of preconditioning time on the voltammetric response was studied for the detection of 100 µM propranolol. The methodology (which is summarized schematically in Figure 2) was that the organogel phase held within the micropores of the silicon membrane was immersed in a beaker containing an artificial saliva aqueous phase and 100 µM propranolol and step 1 was carried out. In step 1, DPV was applied (scanning in the positive direction, from 0.4 to 0.96 V) causing the extraction of propranolol from the artificial saliva aqueous phase into the organogel phase. In step 2, a preconditioning step at a potential of 0.4 V (a potential at which the propranolol should back-extract from the organogel phase into the aqueous phase) was applied for a given time (15, 30, 45, 60, 90, 120, or 180 s). The purpose of this step was to ensure that all the extracted propranolol from step 1 was removed from the gel prior to the Analytical Chemistry, Vol. 81, No. 6, March 15, 2009

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Figure 3. Differential pulse voltammetry investigating the effect of preconditioning times. (A) Step 1: DPV (scanning in the positive direction) for 100 µM propranolol after 15 s of preconditioning time. (B) Step 4: DPV (scanning in the negative direction) in a beaker (just containing artificial saliva) after a preconditioning time of 15 s in step 3. Peak current is observed. (C) Step 1: DPV (scanning in the positive direction) for 100 µM propranolol after 90 s preconditioning time. (D) Step 4: DPV (scanning in the negative direction) in a beaker (just containing artificial saliva) after a preconditioning time of 90 s in step 3. Peak current is not observed.

next experiment. The organogel/silicon membrane was then removed from the beaker in step 3 and was rinsed with deionized water to remove any traces of propranolol on the outer surface. The membrane was then immersed into a solution of artificial saliva aqueous phase without any drug. A DPV scan (scanning in the negative direction, from 0.96 to 0.4 V) as step 4 was then used to check whether any propranolol remained in the organogel phase. If it was present, then it should be detected by the DPV scan as a stripping signal. The DPV scan (step 4) produced peak currents after preconditioning times of 15, 45, and 60 s (see Figure 3B), indicating that propranolol still remained in the gel after step 2, the preconditioning step. Therefore, these times were not sufficient for extensive organogel cleaning. However, preconditioning times of 90, 120, and 180 s (see Figure 3D) were sufficient for extensive organogel cleaning (i.e., after step 4, no peak currents were observed, indicating that no propranolol remained in the gel after step 2). A preconditioning time of 90 s at an applied potential of 0.4 V was therefore chosen as the parameter for organogel cleaning between all subsequent DPV or DPSV experiments. Differential Pulse Voltammetry at the Micro-ITIES Array. As observed previously, CV was capable of detecting propranolol, but in order to detect lower concentrations of the analyte more sensitive voltammetric techniques were necessary. Initially, DPV was employed as the analytical technique. This technique consisted of a preconditioning step and a voltammetric analysis step. The analyte sample was added from a stock solution of propranolol to the artificial saliva aqueous phase. The lowest concentration of propranolol detected was ca. 0.1 µM using backgroundsubtracted DPV, scanning in the positive direction (see Figure 4). A calibration curve was plotted for increasing concentrations of propranolol (see Figure 4 inset). Increase in the concentration of propranolol resulted in the increase of the peak current, with a linear concentration dependence range between 0.1 and 2 µM 2348

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Figure 4. DPV of increasing propranolol concentrations in artificial saliva at the micro-ITIES array. DPV (background-subtracted) (DPVbs) response for 0.1 (light gray), 0.2, 0.5, 0.7, 1, 2, 5, and 10 µM (black) propranolol. Inset: calibration curve of peak current vs concentration.

(ip ) 1.34 (nA µM-1)(concentration) + 0.24 (nA), R ) 0.9759, n ) 7). In order to improve the achieved detection limit of 0.1 µM propranolol, DPSV was assessed as the detection method. Differential Pulse Stripping Voltammetry at the MicroITIES Array. This technique consisted of a preconditioning step, a preconcentration step, and a voltammetric detection step. The preconditioning step was employed for 90 s at a potential of 0.4 V. The influence of the preconcentration time was investigated at a potential of 0.86 V for a 0.5 µM concentration of propranolol. This step caused the accumulative extraction of propranolol into the organogel phase. The preconcentration step is crucial in DPSV because it is this factor which enables lower detection limits to be achieved. The preconcentrated propranolol was then stripped out of the gel using DPSV (background-subtracted). As shown in Figure 5, the stripping peak current increased as the preconcentration time increased. As seen in the calibration curve (inset), at longer preconcentration times a saturation effect occurred where

Figure 5. Influence of preconcentration times on the DPSV (background-subtracted) of 0.5 µM propranolol in artificial saliva at the micro-ITIES. DPSVbs response of 0 (light gray), 5, 15, 30, 45, 60, 90, 120, and 180 s (black) deposition times. Inset: graph of current vs preconcentration time.

Figure 6. Electrochemistry of increasing concentrations of propranolol in artificial saliva at the micro-ITIES using DPSV (backgroundsubtracted). DPSVbs response of 0.05 (light gray), 0.07, 0.09, 0.11, 0.15, 0.17, 0.2, 0.4, 0.6, 0.8, and 1 µM (black) propranolol. Inset: calibration curve of current vs concentration.

maximum propranolol detection current was observed. This saturation effect was also observed for the detection of oligopeptides37 at the micro-ITIES array for increasing preconcentration times. All subsequent experiments employed a preconcentration time of 180 s to improve the limit of detection. Increasing Concentrations of Propranolol. Increasing concentrations of propranolol were added to the artificial saliva and background-subtracted DPSV (DPSVbs) was used as the analytical method. As illustrated in Figure 6, as the concentration of propranolol increased the stripping peak current increased also. There was a linear increase in current at the lower concentrations, between 0.05 and 0.2 µM (ip ) -8.13 (nA µM-1)(concentration) + 0.07 (nA), R ) 0.9929, n ) 7), with a saturation effect occurring at higher concentrations. The lowest concentration detectable in this case was 0.05 µM, in comparison to 0.1 µM

when DPV was employed. The calculated limit of detection, based on 3 times the standard deviation of the blank (3sb) was 0.02 µM. The low detection limit was due to combination of the increased mass-transport rate at the micro-ITIES, the DPSV method, and background subtraction. Improvement of the limit of detection is extremely important in this electrochemical detection technique in order for it to be used as a viable technique for drug detection in saliva. Commercially available drug testing kits (i.e., Toxiquick, Cozart)1 are capable of detecting opiates and other illicit drugs to a concentration of nanograms per milliliter. Propranolol detection at the microITIES array was possible to a concentration of 5.92 ng/mL or 0.02 µM. This concentration compares well with limits of detection previously published for propranolol including 0.2 µM (adsorptive stripping differential pulse voltammetry),50 5 µM (potentiometric membrane electrode),51 0.02 µM (cathodic adsorptive stripping voltammetry),52 and 0.1 µM (capillary electrophoresis).53 CONCLUSION The studies reported in this paper have shown that propranolol can be detected in artificial saliva at the polarized micro-ITIES array at 20 nM concentration. The low limit of detection was possible due to the miniaturized interfaces formed at the mouths of silicon micropores and the preconcentration step used in the DPSV technique. The inclusion of a preconditioning step, which ensures efficient cleaning of the organogel phase prior to each preconcentration in the DPSV method, was crucial to the analytical performance obtained. These results demonstrate that electrochemistry at the ITIES is a viable analytical strategy for the determination of bioactive compounds in realistic sample matrixes when it is combined with suitable devices (micropore membranes in this case) and analytical methodology. ACKNOWLEDGMENT This work was supported by Science Foundation Ireland (Grant No. 07/IN.1/B967). Fabrication facilities employed in this research were supported by the Higher Education Authority’s Programme for Research in Third Level Institutions (PRTLI). Received for review December 15, 2008. Accepted January 25, 2009. AC802644G (50) Radi, A.; Wassel, A. A.; El Ries, M. A. Chem. Anal. 2004, 49, 51–58. (51) El-Tohamy, M.; El-Maamly, M.; Shalaby, A. Bull. Pharm. Sci. 2006, 29, 488–500. (52) Ghoneim, M. M.; Beltagi, A. M.; Radi, A. Quim. Anal. 2002, 20, 237–241. (53) Bai, X. X.; You, T. Y.; Sun, H. W.; Yang, X. R.; Wang, E. K. Electroanalysis 2000, 12, 1379–1382.

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