On-Chip Electro Membrane Extraction with Online Ultraviolet and

Dec 10, 2010 - amitriptyline by rat liver microsomes. For years, liquid-liquid extraction (LLE) has been used for sample preparation prior to chromato...
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Anal. Chem. 2011, 83, 44–51

On-Chip Electro Membrane Extraction with Online Ultraviolet and Mass Spectrometric Detection Nickolaj Jacob Petersen,*,† Sunniva Taule Foss,†,‡ Henrik Jensen,† Steen Honore´ Hansen,† Christian Skonberg,† Detlef Snakenborg,§ Jo¨rg P. Kutter,§ and Stig Pedersen-Bjergaard†,‡ Department of Pharmaceutics and Analytical Chemistry, Faculty of Pharmaceutical Sciences, University of Copenhagen, DK-2100 Copenhagen, Denmark, School of Pharmacy, University of Oslo, Post Office Box 1068 Blindern, 0316 Oslo, Norway, and Department of Micro- and Nanotechnology, Technical University of Denmark, 2800 Lyngby, Denmark Electro membrane extraction was demonstrated in a microfluidic device. The device was composed of a 25 µm thick porous polypropylene membrane bonded between two poly(methyl methacrylate) (PMMA) substrates, each having 50 µm deep channel structures facing the membrane. The supported liquid membrane (SLM) consisted of 2-nitrophenyl octyl ether (NPOE) immobilized in the pores of the membrane. The driving force for the extraction was a 15 V direct current (DC) electrical potential applied across the SLM. Samples containing the basic drugs pethidine, nortriptyline, methadone, haloperidol, loperamide, and amitriptyline were used to characterize the system. Extraction recoveries were typically in the range of 65-86% for the different analytes when the device was operated with a sample flow of 2.0 µL/min and an acceptor flow of 1.0 µL/min. With the sample flow at 9.0 µL/min and the acceptor flow at 0.0 µL/min, enrichment factors exceeding 75 were obtained during 12 min of operation from a total sample volume of only 108 µL. The on-chip electro membrane system was coupled online to electrospray ionization mass spectrometry and used to monitor online and real-time metabolism of amitriptyline by rat liver microsomes. For years, liquid-liquid extraction (LLE) has been used for sample preparation prior to chromatography and electrophoresis. In LLE, target analytes are extracted from an aqueous sample and into an organic solvent immiscible with water. The efficiency of LLE is basically determined by partition coefficients and can easily be predicted on the basis of the molecular structure of the target analytes. LLE is widely used because it is simple, rapid, and efficient and because it gives reasonable sample cleanup. The sample cleanup is obtained as polar and charged compounds, which often dominate as the matrix in aqueous samples, are not extracted into organic solvents of low polarity. In recent years, LLE has been developed and become more sophisticated in different directions. Important directions include the development of supported liquid membrane extraction (SLM),1,2 * Corresponding author. E-mail: [email protected]. † University of Copenhagen. ‡ University of Oslo. § Technical University of Denmark. (1) Audunsson, G. Anal. Chem. 1986, 58, 2714–2723.

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drop-to-drop liquid-phase microextraction,3 and hollow-fiber liquidphase microextraction (LPME).4,5 Another important direction has been the down-scaling and implementation of LLE to microfluidic chip devices, either using tiny droplets of organic solvent6-20 or based on different membrane approaches.21-23 The major incentive for the latter research has been to build micro total analysis systems (µ-TAS) by fully integrating sample preparation with separation and detection on the chip devices. With such systems, chemical analysis can be performed on very small sample volumes, with low consumption of chemicals and reagents, and with fast kinetics. The latter is due to the very short diffusion path in microfluidic chip devices.24 Recently, we demonstrated for the first time on-chip electro membrane extraction (EME).25 This system represented a new way of implementing LLE-like principles to microfluidic chip (2) Jonsson, J. A.; Mathiasson, L. Trends Anal. Chem. 1999, 18, 318–325. (3) Jeannot, M. A.; Cantwell, F. F. Anal. Chem. 1996, 68, 2236–2240. (4) Pedersen-Bjergaard, S.; Rasmussen, K. E. Anal. Chem. 1999, 71, 2650– 2656. (5) Pedersen-Bjergaard, S.; Rasmussen, K. E. J. Chromatogr. B 2005, 817, 3–12. (6) Tokeshi, M.; Minagawa, T.; Kitamori, T. J. Chromatogr. A 2000, 894, 19– 23. (7) Tokeshi, M.; Minagawa, T.; Kitamori, T. Anal. Chem. 2000, 72, 1711– 1714. (8) Burns, J. R.; Ramshaw, C. Lab Chip 2001, 1, 10–15. (9) Hisamoto, H.; Horiuchi, T.; Tokeshi, M.; Hibara, A.; Kitamori, T. Anal. Chem. 2001, 73, 1382–1386. (10) Minagawa, T.; Tokeshi, M.; Kitamori, T. Lab Chip 2001, 1, 72–75. (11) Hibara, A.; Nonaka, M.; Hisamoto, H.; Uchiyama, K.; Kikutani, Y.; Tokeshi, M.; Kitamori, T. Anal. Chem. 2002, 74, 1724–1728. (12) Kerby, M. B.; Spaid, M.; Wu, S.; Parce, J. W.; Chien, R. L. Anal. Chem. 2002, 74, 5175–5183. (13) Surmeian, M.; Slyadnev, M. N.; Hisamoto, H.; Hibara, A.; Uchiyama, K.; Kitamori, T. Anal. Chem. 2002, 74, 2014–2020. (14) Tokeshi, M.; Minagawa, T.; Uchiyama, K.; Hibara, A.; Sato, K.; Hisamoto, H.; Kitamori, T. Anal. Chem. 2002, 74, 1565–1571. (15) Kuban, P.; Berg, J.; Dasgupta, P. K. Anal. Chem. 2003, 75, 3549–3556. (16) Chen, H.; Fang, Q.; Yin, X. F.; Fang, Z. L. Lab Chip 2005, 5, 719–725. (17) Shen, H.; Fang, Q.; Fang, Z. L. Lab Chip 2006, 6, 1387–1389. (18) Xiao, H.; Liang, D.; Liu, G. C.; Guo, M.; Xing, W. L.; Cheng, J. Lab Chip 2006, 6, 1067–1072. (19) Mary, P.; Studer, V.; Tabeling, P. Anal. Chem. 2008, 80, 2680–2687. (20) Shen, H.; Fang, Q. Talanta 2008, 77, 269–272. (21) Maruyama, T.; Matsushita, H.; Uchida, J.; Kubota, F.; Kamiya, N.; Goto, M. Anal. Chem. 2004, 76, 4495–4500. (22) Cai, Z. X.; Fang, Q.; Chen, H. W.; Fang, Z. L. Anal. Chim. Acta 2006, 556, 151–156. (23) Wang, X. Y.; Saridara, C.; Mitra, S. Anal. Chim. Acta 2005, 543, 92–98. (24) Tabeling, P. Introduction to Microfluidics; Oxford University Press: New York, 2005. 10.1021/ac1027148  2011 American Chemical Society Published on Web 12/10/2010

devices. In the system for on-chip electro membrane extraction, the sample solution, which contained different nonpolar basic drugs as model analytes, was pumped into the sample channel of the chip. From this channel, the model analytes were extracted through a hydrophobic supported liquid membrane (SLM) and into an acceptor reservoir in the chip located on the other side of the SLM. The driving force for the extraction was a DC electrical potential, where the positive electrode (anode) was placed in the sample channel and the negative electrode (cathode) was placed in the acceptor reservoir. The aqueous media on both sides of the SLM were acidic; the basic drugs were therefore protonated, and they migrated across the SLM under the influence of the electrical field. The purpose of the SLM was to provide a hydrophobic barrier between the sample and the acceptor, and this resulted in very efficient cleanup of urine samples. Thus, when tuned for nonpolar basic drugs, matrix components like small inorganic salts, neutral compounds, acidic compounds, and polar compounds were discriminated by the SLM. The purpose of the electrical potential was to increase the distribution of the basic analytes into the membrane on the sample side and out of the membrane on the acceptor side.26 Because the sample residence time in the chip was only a few seconds, rapid migration of the analytes into the SLM was crucial to obtain high extraction efficiency, and this was accomplished by using shallow channels (short diffusion distances). In addition, the electrical potential served as an adjustable driving force for the extraction, as both the magnitude and the direction was easily and rapidly adjusted by the power supply. In our recent on-chip electro membrane extraction system,25 the acceptor solution was stagnant and after each extraction, the acceptor solution was removed manually by a pipet and analyzed offline by capillary electrophoresis. In the present work, we demonstrate a new approach by introducing a flow also in the acceptor channel. With this dynamic system, the acceptor solution is continuously pumped into a UV detector or a mass spectrometer for online analysis. This new and dynamic on-chip electro membrane extraction system is optimized with respect to operational parameters. The influence of sample flow rate and the dimensions of the device are discussed relative to a theoretical model developed for the system. A potential application is demonstrated by online and real-time mass spectrometric measurement of the metabolism of amitriptyline by rat liver microsomes. EXPERIMENTAL SECTION Chemicals and Sample Solutions. Pethidine hydrochloride, nortriptyline hydrochloride, methadone hydrochloride, haloperidol, loperamide hydrochloride, and amitriptyline hydrochloride were all obtained from Sigma-Aldrich (St. Louis, MO). 2-Nitrophenyl octyl ether (NPOE), 2-nitrophenyl pentyl ether, 2,4dimethyl-1-nitrobenzene, and 1-ethyl-2-nitrobenzene where obtained from Fluka (Buchs, Switzerland). Rat liver microsomes (male Sprague-Dawley, pooled; 20 mg/mL) were obtained from BD Biosciences (San Jose, CA), and β-nicotinamide adenine dinucleotide 2′-phosphate reduced tetrasodium salt hydrate (NADPH) was obtained from Sigma-Aldrich. (25) Petersen, N. J.; Jensen, H.; Hansen, S. H.; Foss, S. T.; Snakenborg, D.; Pedersen-Bjergaard, S. Microfluid. Nanofluid. 2010, 9, 881–888. (26) Petersen, N. J.; Jensen, H.; Hansen, S. H.; Rasmussen, K. E.; PedersenBjergaard, S. J. Chromatogr. A 2009, 1216, 1496–1502.

Figure 1. Schematic illustration of on-chip electro membrane system.

A stock solution containing 1 mg/mL each of pethidine, nortriptyline, methadone, haloperidol, and loperamide was prepared in 10% (v/v) ethanol and stored protected from light at 4 °C. Sample solutions containing the five compounds were prepared by dilution of the stock solution either with 10 mM HCl, 100 mM formic acid, and 50 mM phosphate buffer, pH 7.4, or with human urine with 50% (v/v) 100 mM formic acid (providing a spiked urine sample). HCl (10 mM) or formic acid (100 mM) was used as the acceptor solution. For the metabolism experiment, stock solutions of 5 mM amitriptyline, 10 mM NADPH, 100 mM MgCl2, and 1.0 M potassium phosphate buffer (pH 7.4) were prepared individually in deionized water. Preparation of Electro Membrane Chip. The electro membrane extraction chip is schematically illustrated in Figure 1. Each electro membrane chip contained five individual channels for extraction and was composed of two poly(methyl methacrylate) (PMMA) (53 × 53 × 2.1 mm3) plates. In both plates, the 6 mm long sample and acceptor channels (rectangular) with a depth of 50 µm and a width of 2.00 mm were milled on a CNC micromilling machine (Folken M3400 E CNC mini mill, Folken Industries, Glendale, CA). At both ends of the channels, 1.6 mm i.d. holes were drilled through the plate to serve as inlet and outlet for the sample solution. Above the sample channels, a porous polypropylene membrane was placed that had 25 µm thickness, 55% porosity, and 0.21 × 0.05 µm pores (Celgard 2500 microporous membrane; Celgard, Charlotte, NC). The second PMMA plate was placed above the membrane and aligned to match the positions of the sample and acceptor channels, and the whole assembly was fixed by solvent-assisted bonding with ethanol and cured in a 70 °C oven. The sample and acceptor solutions were delivered by two separate syringe pumps (KDS-100-CE, kdScientific, Holliston, MA), each operated with a 1000 µL gastight 1001 syringe (Hamilton, Bonaduz, Switzerland). The diameter of the access holes to the sample and acceptor channels (1.6 mm i.d.) allowed for almost zero dead volume and a leaktight connection when 1/16in. o.d. poly(tetrafluoroethylene) (PTFE), poly(ether ether ketone) (PEEK), or steel tubing was inserted directly in the holes. The syringe containing the acceptor solution was connected to the inlet reservoir with 1/16-in. o.d. stainless steel HPLC tubing, which also served as the cathode for the electro membrane extraction. The electric contact for the sample channel (anode) was supplied from a 0.076 mm o.d. platinum wire (Sigma-Aldrich) inserted into a 2 cm long 1/16-in. o.d. PTFE tube placed in the sample outlet reservoir. The electrodes were connected to a variable DC power supply (EL302T Triple power supply, Thurlby-Thandar Instruments Ltd., CamAnalytical Chemistry, Vol. 83, No. 1, January 1, 2011

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bridgeshire, U.K.). The power supply allowed the cathode to be operated at ground (0 V), which had the advantage that the acceptor solution and electrospray interface were grounded when operating the device was operated in electrospray ionization mass spectrometry (ESI-MS) mode. When coupled online to the UV or MS detection, a 375 µm o.d. fused silica capillary (Polymicro Technologies, Phoenix, AZ) was connected to the acceptor outlet reservoir. The fused silica capillary was inserted in a 1/16-in. o.d. PEEK tubing sleeve (F230, Upchurch Scientific, Oak Harbor, WA) to make the capillary fit the 1.6 mm i.d. hole. The high flow resistance of this connection capillary made it necessary to seal the connections to the device with hot-melt adhesive [glue stick universal (ethylene-vinyl acetate copolymer based), Bostic Nordic, Helsingborg, Sweden]. Prior to connection of the tubing to the device, 2-nitrophenyl octyl ether (NPOE) was immobilized in the membrane. This was achieved by loading 0.2 µL of NPOE directly onto the membrane, from one of the reservoirs, by use of a Eppendorf Research pipet (Eppendorf, Hamburg, Germany). The successful filling of the membrane separating the sample and acceptor channel could be observed visually because the membrane changed from a white appearance to transparent when filled with NPOE. Subsequently, the tubing for the sample and acceptor flow were connected to the reservoirs and fixed with the hot-melt adhesive. Samples were either filled directly into the sample syringe and delivered continuously to the chip or were injected into the system by a 6-port valve with a 20 µL loop (Valco Instruments Co. Inc., Houston, TX) inserted between the sample syringe pump and the chip. In the latter case, the sample syringe was filled with either 10 mM HCL or 100 mM formic acid. Analysis of the sample extract was either done online, by coupling to an ESI-MS interface or UV detector, or done offline, by capillary electrophoresis (CE)-UV analysis. The offline CE analysis required the collection of a minimum 7 µL of acceptor phase to fill the CE sample vial to the minimum level required for injection. Capillary Electrophoresis. Capillary electrophoresis (CE) was performed with an Agilent Technologies HP3D CE instrument (Agilent Technologies, Waldbronn, Germany) equipped with a UV detector operated at 200 nm. The running buffer was 25 mM sodium dihydrogen phosphate adjusted to pH 2.7 with orthophosphoric acid. Separations were performed at 20 kV in a 75 µm i.d. fused-silica capillary (TSP075375, Polymicro Technologies, Phoenix, AZ) with an effective length of 24.5 cm. Online Measurements with UV Detection. Online measurements were conducted with the UV detector of the Agilent Technologies HP3D CE instrument. The acceptor channel outlet of the chip was connected with a 75 µm i.d. fused silica capillary (TSP075375, Polymicro Technologies), and this capillary was coupled to the CE UV detector. The required length of the capillary from the chip to reach the UV detector was 34 cm in normal configuration, but by drilling a new access hole for the capillary in the CE instrument this length was successfully shortened to 17 cm, corresponding to a volume of only 0.75 µL. Operating with an acceptor flow rate of 3 µL/min, this volume introduced a delay of 15 s from the extraction cell to the UV detector. 46

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Online Measurements with Electrospray Ionization Mass Spectrometry. An in-house-built nanospray interface was used for the ESI-MS coupling. A 50 µm i.d. fused silica capillary (TSP050375, Polymicro Technologies) was sharpened by first pulling the end of the capillary in a flame, and subsequently the spray tip was etched in 48% hydrofluoric acid (Riedel-de Hae¨n, Seelze, Germany) while air was blown through the capillary. The etching assured that the capillary maintained its 50 µm i.d. at the spray tip, thereby minimizing the possibility of clogging. The capillary was cut to a total length of 15 cm and inserted in the outlet reservoir of the acceptor channel via a 1/16-in. o.d. PEEK tubing sleeve (F-230, Upchurch Scientific) and fixed with the hot-melt adhesive. The spray capillary was inserted into an inhouse-built CE-ESI-MS coaxial interface mounted on an XYZ stage directly in front of the MS entrance (Bruker-Esquire LCMS ion trap, Bruker Daltronics Inc., Billerica, MA). The acceptor solution used for the online coupling to the MS was 100 mM formic acid delivered at a flow rate of 0-6 µL/min. The high voltage for the ESI was supplied from the MS instrument by operating the end plate at the MS entrance at -4.5 kV while the spray capillary was grounded at the acceptor channel. The sharpened spray capillary provided a stable electrospray even without the application of a nebulizer gas, but the sensitivity was slightly improved when the spray was operated with a coaxial nebulizer gas (nitrogen) operated at a pressure of 50 psi. The volume of the 15 cm spray capillary was only 0.3 µL. Operating with an acceptor flow rate of 3 µL/ min corresponded to a delay of only 6 s from the extraction device. Metabolism of Amitriptyline by Rat Liver Microsomes. The metabolism was performed by preparing a reaction solution of rat liver microsomes (RLM) and amitriptyline from the stock solutions to a final composition of 1 mg/mL RLM, 50 µM amitriptyline, and 5 mM MgCl2 in 100 mM potassium phosphate (pH 7.4). The metabolism was initiated by adding the cofactor NADPH to the reaction mixture by a pipet to obtain a concentration of 1 mM NADPH. The metabolism was performed online in a 1000 µL gastight 1001 syringe (Hamilton) temperature-controlled at 37 °C by circulating water around the syringe in an in-house-built device. The syringe was connected to the EME chip by 8 cm long PEEK tubing (175 µm i.d., 1/16-in. o.d., Upchurch Scientific) having a total dead volume of 2 µL. The syringe pump continuously pumped the reaction solution to the chip at a flow rate of 9 µL/min. Calculation of the Enrichment Factor and Recovery. The enrichment factor (EFi) for the analyte i was calculated according to eq 1:

EFi )

Caioutlet Csiinlet

(1)

where Caioutlet is the concentration of the analyte i at the outlet of the acceptor channel and Csiinlet is the initial sample concentration of the analyte at the sample inlet reservoir. The recovery was defined as the fraction of analyte i in the sample solution that was transferred to the acceptor phase. When the extraction device was operated with bulk flow rates of va and vs for the acceptor and sample solution, respectively, the

recovery (Ri) for the individual analytes i was calculated according to eq 2:

Ri )

vaCaioutlet vsCsiinlet

)

va EF vs i

(2)

The concentration, Caioutlet, of analyte in the acceptor solution was estimated by CE separations of the extract. The standard curves were prepared by CE separations of different concentrations of the analytes prepared directly in the acceptor solution and without any extraction/concentration. For online monitoring with UV and MS detection, the concentration was based on the signal height, and standard curves had been made for the analyte signal for different analyte concentrations in acceptor solution. For stopped-flow experiments, only the enrichment factor was estimated on the basis of the increase in signal height. Calculations based on eq 2 were only performed from experiments with flow in the acceptor channel. RESULTS AND DISCUSSION The basic setup for the double-flow on-chip EME system is illustrated in Figure 1. Pethidine, nortriptyline, methadone, haloperidol, and loperamide were selected as principal model analytes for the study. The reason for this selection was that the same compounds had been extracted in our recent on-chip EME system with stagnant acceptor solution, and this enabled the new doubleflow system to be compared with this previous configuration.25 The model analytes were all basic drugs of relatively low polarity. The sample solution consisted initially of the five model analytes dissolved at 10 µg/mL in 10 mM HCl. HCl served as background electrolyte in the sample, to ensure that the model analytes were protonated. The sample solution was pumped into the chip with a flow rate in the range of 1.75-12.0 µL/min, and the acceptor solution was pumped into the chip with a flow rate of 0.25-3.0 µL/min. The ratio between the sample and acceptor flow rate strongly influenced the enrichment factor, and in some cases the flow was even stopped during extraction (stop-flow mode) to achieve the highest possible enrichment factors. The DC voltage applied across the SLM served as the driving force for the extraction, and was in the range from -2 to +30 V. The acceptor solution was collected at the acceptor channel outlet and analyzed offline by capillary electrophoresis to obtain accurate quantitative information of the extraction of the five analytes at different flow rates and extraction voltages. Investigation of Operational Parameters. In a first experiment, the system performance was characterized with different flow rates for the sample solution. The sample flow rate was varied within the range 1.75-4.0 µL/min while the acceptor flow rate was constant at 1.0 µL/min (Figure 2). Recoveries were found to be approximately constant at the lowest flow rates and started to decrease at flow rates higher than 2.0-3.0 µL/min. As the flow rate increased, the average residence time in the sample channel decreased and was approximately 16 s for the flow rate at 3.0 µL/ min. The recoveries obtained with the present double-flow system were superior to earlier results published on a similar on-chip system operated with the same sample flow rates but with a stagnant acceptor solution.25 The recoveries obtained with a sample flow rate of 3.0 µL/min in the current design corresponded

Figure 2. Extraction recoveries versus flow rate of sample. SLM ) 2-nitrophenyl octyl ether (NPOE); sample ) 10 mM HCl containing the five drugs each at 10 µg/mL; acceptor ) 1.0 µL/min of 10 mM HCl; voltage ) 15 V; extraction time ) 7 min. Recoveries were determined by off-chip CE separations.

to 80% for nortriptyline, as compared to only 34% in the previous work.25 Similarly, the recovery for all the other analytes also increased: methadone from 41% to 85%, haloperidol from 37% to 79%, loperamide from 52% to 86%, and pethidine from 12% to 52%. The influence of different flow rates of the acceptor solution was characterized in the range 0.25-2.0 µL/min, while the sample flow rate was kept constant at 3.0 µL/min. The recoveries were almost independent of the acceptor flow rate and were at the 80% level for nortriptyline, methadone, haloperidol, and loperamide and at the 45% level for pethidine. The results showed that the acceptor flow rate had no significant influence on the recovery for operation in the range 0.25-2.0 µL/min. The higher recovery in the present layout as compared to the previous design with the stagnant acceptor solution is probably due both to the use of flow conditions in the acceptor channel and to a larger area of the SLM in contact with the acceptor solution. In the present layout the acceptor solution had a contact area along the whole sample channel corresponding to 16 mm2, compared to a contact area of only 3.14 mm2 in the previous design, where the area was determined by the diameter of the acceptor reservoir. Enrichment factor as a function of the acceptor flow rate is illustrated in Figure 3. At 0.25 µL/min flow for the acceptor solution, four of the model analytes were preconcentrated by a factor of 9.6-10.2 from 21.0 µL of sample (delivered at 3.0 µL/ min). This illustrated that preconcentration easily can be adjusted by modifying the ratio between the sample and the acceptor flow rates. Another interesting feature of the double-flow on-chip EME system was observed when the extraction voltage was optimized for the respective model analytes (Figure 4). In this case, the voltage was varied between -2 and 20 V, and essentially all recoveries increased as the voltage was increased from 0 to 10 V, whereas it remained unaffected as the voltage was increased from 10 to 20 V. Thus, only 10 V was required as the potential for efficient extractions. However, it was decided to operate the extraction device at 15 V to ensure that any variations in the sample conductivity did not affect the system performance. At 0 V the recovery of all the analytes was less than 1%; this passive extraction could be completely suppressed by applying a voltage of -2 V. A practical consequence of the low voltage and the high Analytical Chemistry, Vol. 83, No. 1, January 1, 2011

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In addition, for the acceptor solution, 10 mM HCl was replaced by 100 mM formic acid. The reason for using 100 mM formic acid was to test an acceptor solution compatible with electrospray ionization mass spectrometry (ESI-MS). As illustrated in Table 2, 100 mM HCOOH also worked efficiently as acceptor solution. Theoretical Considerations. Recoveries in dynamic on-chip EME were modeled on the basis of previous derivation of conversion efficiencies in electrochemical flow reactors.27 With the assumptions of a laminar flow profile in the sample solution, that the recoveries are controlled by mass transport in the sample solution, and that no accumulation of analyte occurs within the SLM, then the extraction recovery, Ri, for an analyte i can be described as27 Figure 3. Extraction enrichment versus flow rate of acceptor. SLM ) 2-nitrophenyl octyl ether (NPOE); sample ) 3.0 µL/min of 10 mM HCl containing the five model drugs at 10 µg/mL; acceptor ) 10 mM HCl; voltage ) 15 V; extraction time ) 7 min. Recoveries were determined by off-chip CE separations.

Ri ) 1 -

Csioutlet Csiinlet

) 1 - e-am0i/vs

(3)

where Csioutlet is the concentration of analyte at the outlet of the sample channel, m0i is the mass transfer coefficient, a is the contact area with the SLM, and vs is the volumetric flow rate of sample. In electrochemical flow reactors, the following equation may be used to describe m0i:27,28 m0i ) biUR

(4)

where U is the linear flow velocity of sample, R is a constant dependent on mass transport characteristics, and bi is an empirical constant dependent on the specific analyte and flow channel geometry. Combining eqs 3 and 4 gives the following equation: Figure 4. Extraction recoveries versus extraction voltage. SLM ) 2-nitrophenyl octyl ether (NPOE); sample ) 3.0 µL/min of 10 mM HCl containing the five drugs each at 10 µg/mL; acceptor ) 1.0 µL/ min of 10 mM HCl; extraction time ) 7 min. Recoveries were determined by off-chip CE separations. All extractions were performed on the same device over two separate days in random order. Error bars reflect the overall standard deviation, SD (n ) 3 or 6).

electric resistance across the membrane was that the current in the system was below 1-2 µA in all cases. Electrolysis at the electrodes was therefore almost absent, and the pH on both sides of the SLM remained unaffected.25 The double-flow on-chip EME system was also characterized with different organic solvents as the SLM. The results are summarized in Table 1. As seen from the results, both 2-nitrophenyl octyl ether (NPOE), 2-nitrophenyl pentyl ether, 2,4dimethyl-1-nitrobenzene, and 1-ethyl-2-nitrobenzene worked as SLM in the double-flow on-chip EME system. Superior results were obtained with NPOE, and this solvent was used throughout this study. In a final experiment in this section, alternative background electrolytes were tested in the sample and in the acceptor solution. For the sample solution, 10 mM HCl was replaced with respectively 100 mM HCOOH (pH 2) and 100 mM potassium phosphate buffer, pH 7.4. The latter was tested to simulate physiological conditions. At pH 7.4, the model analytes were only partly protonated and therefore also extracted by the pH gradient across the SLM. As illustrated in Table 2, the three different background electrolytes in the sample provided very high recoveries at 15 V. 48

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R-1/A

Ri ) 1 - e-abi(U)

(5)

where A is the cross-sectional area of the sample channel perpendicular to the flow direction. If a rectangular sample channel is assumed and U is expressed from vs, we can rewrite eq 5 as R-1/dR

Ri ) 1 - e-biL(vs /w)

(6)

where d is the depth of the sample channel and L is the length of the SLM having the same width, w, as the sample channel. From eqs 5 and 6 it is seen that the recoveries depend on the geometrical configuration of the sample channel, in the way that a wide and long SLM is beneficial. In Figure 2 the extraction recoveries versus sample flow rate are plotted and compared with the theoretical recoveries based on eq 6, using the geometrical parameters of the chip and a literature value of R ) 1/3 based on the Levich equation28 and b ) 0.0008 m/min. The theoretical lines are plotted in Figure 2 for channel depths of 25, 50, and 75 µm to illustrate the effect of variations in the channel depth. The theoretical recoveries were in accordance with the experimental results for nortriptyline, methadone, and haloperidol. Pethidine had a lower extraction recovery corresponding to a b value of approximately 0.000 35 m/min. The expected dependence of the (27) Bard, A. J.; Faulkner, L. R. Electrochemical Methods: Fundamentals and Applications, 2nd ed.; John Wiley & Sons, Inc.: New York, 2001. (28) Cooper, J. A.; Compton, R. G. Electroanalysis 1998, 10, 141–155.

Table 1. Recoveries with Different Organic Solvents as Supported Liquid Membranea recovery (%) pethidine

nortriptyline

methadone

haloperidol

loperamide

50 65 55 44

74 69 62 50

75 66 63 52

73 67 59 48

70 68 63 54

2-nitrophenyl octyl ether (NPOE) 2-nitrophenyl pentyl ether 2,4-dimethyl-1-nitrobenzene 1-ethyl-2-nitrobenzene

a Sample ) 3.0 µL/min of 10 mM HCl containing a mixture of the five drugs each at 10 µg/mL; acceptor ) 1.0 µL/min of 10 mM HCl; voltage ) 15 V; extraction time ) 7 min.

Table 2. Recoveries Obtained with Different Background Electrolytes in the Sample and in the Acceptor Solutiona % recovery (SD, n ) 3-6) pethidine

nortriptyline

methadone

haloperidol

loperamide

Donor Phaseb 10 mM HCl 100 mM HCOOH 100 mM phosphate (pH 7.4)

61 (4) 22 (6) 45 (11)

89 (3) 66 (14) 70 (12)

94 (3) 81 (14) 82 (9)

86 (4) 64 (14) 73 (10)

96 (4) 90 (14) 78 (8)

Acceptor Phasec 10 mM HCl 100 mM HCOOH

61 (4) 49 (3)

89 (3) 94 (2)

94 (3) 97 (2)

86 (4) 93 (4)

96 (4) 103 (5)

a 2-Nitrophenyl octyl ether (NPOE) used as supported liquid membrane (SLM). Sample flow ) 3.0 µL/min; acceptor flow ) 1.0 µL/min; voltage ) 15 V; extraction time ) 7 min. b With acceptor phase ) 10 mM HCl. c With donor phase ) 10 mM HCl.

channel depth, illustrated by the theoretical lines in Figure 2 for depths of 25, 50, and 75 µm, shows only a minor dependence for the given flow rates. The influence of variations in the channel depth is expected to be even smaller for a longer SLM and lower sample flow rates. At a flow rate of 3.0 µL/min, eq 6 also predicts that the present layout with the 6 mm long SLM is expected to have an recovery approximately 2.2 times higher compared to our previous work,25 where the SLM was limited to approximately 2 mm in the stagnant acceptor solution (assuming b ) 0.0008 m/min in both designs). This is in good accordance with the experimentally observed increase in recoveries obtained with the longer SLM. For nortriptyline R increased 2.3 times; for methadone and haloperidol the increase was 2.1 times; and loperamide increased 1.6 times. For pethidine, where the mass transfer coefficient was smaller (b ) 0.000 35 m/min), the increase in recovery was expected to be approximately 2.6 times, but the actual recovery increased 4.3 times. Future work will address a more in-depth theoretical description of the recovery versus flow rate and its dependence on physical chemical parameters of the analytes. Online Measurements with UV Detection: Preconcentration in Stop-Flow Mode. In a next series of experiments, the acceptor outlet from the on-chip EME system was coupled online to a UV diode-array detector operated at 200 nm. A loop injector was inserted in the sample flow prior to the chip. A syringe pump continuously pumped 9.0 µL/min of 10 mM HCl through the loop injector and into the sample channel of the on-chip EME system. Samples spiked with methadone at 1, 2, 5, 10, and 20 µg/mL in 10 mM HCl were injected into the carrier flow. The injectionloop volume was 20 µL, which at a sample flow rate of 9.0 µL/ min corresponded to a total sample residence time in the extraction chip of 2.2 min. The acceptor solution consisted of 10 mM HCl delivered at a flow rate of 3.0 µL/min. The UV signal of the sample after extraction detected at 200 nm was linearly related to the concentration of methadone in the original sample for the

range 1-10 µg/mL with r2 values of 0.996 and 0.998 for, respectively, the peak height and the area. Repeatability measured as the relative standard deviation (RSD) from six replicate EME-UV experiments was 5% at the 1 µg/mL level and 3% at 10 µg/mL. The limit of detection, defined as the signal height corresponding to 3 times the noise level, was calculated to be 0.54 µg/mL for methadone. In a next series of experiments, the flow in the acceptor channel was stopped during extraction to test the potential for high analyte up-concentration. In this stop-flow mode, mainly the volume of the acceptor solution inside the acceptor channel will determine the maximum achievable concentration factor. The sample consisted of 0.3 µg/mL methadone in 10 mM HCl, and the sample was pumped into the system with a flow of 9.0 µL/ min for 2, 3, 4, 5, 7, and 12 min. After extraction, the acceptor flow (3.0 µL/min) was initiated to transfer the concentrated sample to the UV detector. The peak width measured at half height when the concentrated samples were passing the UV detector was only 0.2 min, corresponding to sample volume of 0.6 µL. Both the area and the peak height of the concentrated peak increased linearly with the concentration time (r2 ) 0.991 and 0.974). The concentration factor of the methadone, based on the increase in peak height, was approximately 27 times for the 4 min extraction and 76 times for the 12 min extraction. Online Measurements with Electrospray Ionization Mass Spectrometry: Extraction of Methadone from Urine. The double-flow on-chip EME system was also connected directly to electrospray ionization mass spectrometry (ESI-MS). The setup was identical for the experiments above, except that the UV detector was replaced by ESI-MS. A syringe pump continuously pumped 9.0 µL/min of 100 mM formic acid through the loop injector and into the sample channel of the on-chip EME system. Different urine samples spiked with methadone at 0.1, 0.25, 1.0, 2.0, 5.0, and 10.0 µg/mL were injected into the carrier flow. The injection-loop volume was 20 µL, which at a sample flow rate of Analytical Chemistry, Vol. 83, No. 1, January 1, 2011

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Figure 5. Mass spectrometry signals for amitriptyline and metabolites versus metabolic reaction time. SL) ) 2-nitrophenyl octyl ether (NPOE); sample flow ) 9.0 µL/min; acceptor ) 3.0 µL/min of 100 mM formic acid; voltage ) 15 V. Amitriptyline (m/z 278), 10hydroxyamitriptyline (m/z 294), nortriptyline (m/z 264), and 10hydroxynortriptyline (m/z 280) are shown.

9.0 µL/min corresponded to a total sample residence time in the extraction chip of 2.2 min. The acceptor solution consisted of 100 mM formic acid delivered at a flow rate of 3.0 µL/min. The MS signal for methadone measured at m/z 310 was linearly related to the concentration in the original sample in the range 0.1-10.0 µg/mL with an r2 value of 0.995. The limit of detection, defined as the signal corresponding to 3 times the noise level, was calculated to be 40 ng/mL for methadone in urine. This type of direct measurement of methadone in urine was possible only after on-chip EME, as the EME process desalted the sample and removed most other matrix components as well. Without on-chip EME, the MS signal at m/z 310 was totally suppressed by the urine matrix. Real-Time Monitoring of Amitriptyline Metabolism by On-Chip Electro Membrane Extraction with Electrospray Ionization Mass Spectrometry. In a final experiment, the onchip EME system was coupled online to ESI-MS and utilized to study the metabolism of amitriptyline by rat liver microsomes. The metabolic reaction occurred in a microsyringe filled with a suspension of rat liver microsomes, MgCl2, potassium phosphate buffer (pH 7.4), amitriptyline as substrate, and NADPH as cofactor. The microsyringe was placed inside a larger plastic syringe, which served as a water bath to maintain a temperature of 37 °C for the metabolic reaction. The outlet of the microsyringe was connected to the inlet of the on-chip EME system. The metabolic reaction was initiated by introducing the cofactor NADPH to the reaction mixture, and at the same time, the microsyringe was started to deliver the reaction mixture continuously to the on-chip EME system with a flow rate of 9.0 µL/min. Amitriptyline and the metabolites were extracted with 15 V across NPOE as SLM and into an acceptor solution of 100 mM HCOOH, which was delivered at a flow rate of 3.0 µL/min. The acceptor flow was directed into the ESI-MS system for continuous real-time monitoring of the metabolism. The result from one of the experiments is shown in Figure 5, where the MS signals of amitriptyline and some of the metabolites are plotted as a function of reaction time. The first minute of the metabolism was not monitored since this time was used to mix the NADPH with the reaction mixture in the syringe and to initiate 50

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the flow rate toward the EME device. As seen from Figure 5, the concentration of amitriptyline decreased during the experiment as measured at m/z 278, and followed apparent first-order kinetics. The half-time for amitriptyline was estimated to be 4.75 min on the basis of five replicate measurements (RSD ) 5.7%). The phase I metabolites nortriptyline at m/z 264 and 10-hydroxyamitriptyline at m/z 294 increased rapidly during the initial phase of the experiment, whereas the concentration of the phase I metabolite 10-hydroxynortriptyline at m/z 280 developed more slowly. After 22 min, the experiment was terminated. Some minor metabolites with m/z 296 and 310 were also detected, which has been observed earlier for amitriptyline.29-31 The on-chip EME system was important for the success of the online experiment. First, the SLM served as an online filter to prevent particulate matter from the rat liver microsome suspension from entering the ESI-MS system. This particulate matter was found not to clog the channels in the chip. Second, the hydrophobic nature of the SLM prevented the salts present in the reaction mixture from entering the ESI-MS system. Even though the sample mixture contained 100 mM sodium phosphate, no sodium adducts were observed in the MS spectra from the acceptor solution. The continuous online desalting of the sample prior to ESI-MS was also important in order to avoid quenching of the MS signal. Technical Considerations. Immobilization of the supported liquid membrane (SLM) was accomplished as described in the Experimental Section. In the double-flow configuration, the SLM was found to be very stable, and the same SLM was typically used for 2-3 days of continuous use without any loss of performance. After this period of time, we shifted to a new channel in the same chip, where a new SLM was immobilized, or to a new channel in another chip. The channel-to-channel or chip-to-chip variability of extraction recoveries was typically in the range of 5-20% RSD. As seen in Figure 5, the time delay from the onset of the metabolism reaction until amitriptyline was detected with maximum response in the MS was approximately 60 s. The majority of this time delay was due to introduction of the cofactor NADPH and onset of the syringe pump. After on-chip EME, the residence time in the connecting electrospray capillary was only 5 s. The outlet tubing from the EME chip was subject to special considerations. Basically, the length of the tubing was determined by the configurations of the UV and MS detectors. The internal diameter was selected as a compromise between avoiding excessive band broadening and avoiding high back-pressure in the chip. A 60 s response time was acceptable in this preliminary work with drug metabolism, but work is in progress to develop a more integrated chip with less response time. CONCLUSIONS AND FUTURE PERSPECTIVES The present work has for the first time demonstrated dynamic on-chip electro membrane extraction, where both the sample and the acceptor solution were delivered continuously to the chip by microsyringe pumps. With the flow of acceptor solution turned on, the system enabled continuous extraction and online measure(29) Bahr, C. V. Xenobiotica 1972, 2, 293–305. (30) Kruger, R.; Holzl, G.; Kuss, H. J.; Schefold, L. Psychopharmacology 1986, 88, 505–513. (31) Wen, B.; Ma, L.; Zhu, M. S. Chem.-Biol. Interact. 2008, 173, 59–67.

ment with UV or MS detection. By reducing the flow of acceptor solution relative to the flow of sample, the system enabled significant preconcentration. The flow of acceptor solution was even turned off, and the system enabled more than 75 times analyte enrichment from 108 µL of sample. The supported liquid membrane (SLM) separating the sample and acceptor played a key role in the system, as the SLM prevented particulate matter, salts, and many other matrix components from entering the acceptor solution. Thus, the SLM provided very efficient sample cleanup from complex biological fluids. This was especially important for online coupling to MS, to avoid serious quenching of the MS signals. The potential of this new type of technology was briefly demonstrated with a continuous and real-time online measurement of the metabolism of amitriptyline by rat liver microsomes.

Compared with hollow-fiber LPME,4,5 the current system offer the possibility of continuous extraction and online measurement. In addition, the current system represents a further downscaling of hollow-fiber LPME and of traditional SLM extraction,1,2 which is beneficial for the handling and extraction of very small sample volumes. Thus, with on-chip electro membrane extraction, analytes can easily be enriched from low-microliter volumes of sample in very short time. However, more work is required in the future to develop and characterize the technique and to develop key applications. Key applications will address basic and acidic drugs and peptides, which have been extracted previously by conventional electro membrane extraction. Received for review June 16, 2010. Accepted November 19, 2010. AC1027148

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