Anal. Chem. 2008, 80, 481-490
Kinetic Calibration Using Dominant Pre-equilibrium Desorption for On-Site and in Vivo Sampling by Solid-Phase Microextraction Simon Ningsun Zhou, Wennan Zhao, and Janusz Pawliszyn*
Department of Chemistry, University of Waterloo, Waterloo, Ontario, Canada N2L 3G1
A new kinetic calibration was developed using dominant pre-equilibrium desorption by solid-phase microextracton. The calibration was based on isotropism between absorption and desorption, which was proved theoretically and experimentally in an aqueous solution and semisolid matrix. This approach allows for the calibration of absorption using desorption to compensate for matrix effects. Moreover, concentration profiles are initially proposed to verify isotropism between the absorption and desorption, while providing a linear approach to obtain time constants for the purpose of quantitative analysis. This linear approach is more convenient, robust, and accurate than the nonlinear version with the previously used time profiles. Furthermore, the target analytes are used as the internal standards; thus, radioactive or deuterated internal standards are not necessary. In addition, dominant preequilibrium desorption utilizes the pre-equilibrium approach and offers a shorter sample preparation time, which is typically suitable for in vivo sampling. This kinetic calibration method was successfully applied to prepare samples of polycyclic aromatic hydrocarbons in a flowthrough system and in vivo pesticide sampling in a jade plant (Crassula ovata). Solid-phase microextraction (SPME) is a sampling and sample preparation technique first invented by Pawliszyn over one decade ago.1 SPME offers many advantages over conventional sampling methods by combining sample extraction, concentration, and introduction into a single step. It has found numerous applications in many disciplines.2 To date, the most well-established and widely used quantification method using SPME is the equilibrium extraction method. In this approach, a partitioning equilibrium between the sample matrix and extraction phase is reached. The extracted amount of analyte by fiber, ne, is given by,3
KfsVfVs ne ) C Vs + KfsVf 0
(1)
where Kfs is the distribution coefficient, Vf is the volume of the fiber coating, Vs is the volume of sample matrix, and C0 is the * To whom correspondence should be addressed. Tel.: +1-519-888-4641. Fax: +1-519-746-0435. E-mail:
[email protected]. (1) Belardi, R. G.; Pawliszyn, J. J. Water Pollut. Res. Can. 1989, 224, 179-184. (2) Pawliszyn, J., Ed. Applications of Solid-Phase Microextraction; Royal Society of Chemistry: Cambridge, U.K., 1999. (3) Pawliszyn, J. Solid-Phase Microextraction-Theory and Practice; Wiley-VCH: New York, 1997. 10.1021/ac701871q CCC: $40.75 Published on Web 12/20/2007
© 2008 American Chemical Society
initial concentration of the analyte in the sample matrix. The volume of a sample matrix is typically large. When the product of Kfs and Vf is essentially negligible compared to Vs, eq 1 can be simplified to
ne ) KfsVfC0
(2)
Alternatively, short-time pre-equilibrium extraction can be used. If convection and agitation are constant in the sample, then the amount of analyte extracted is related to the extraction time. Quantification can then be performed based on the timed accumulation of analytes in the coating. This pre-equilibrium approach was first developed by Ai,4,5 who proposed a dynamic SPME model based on a diffusion-controlled mass-transfer process. On the basis of this process, Chen et al. demonstrated an isotropic behavior between absorption and desorption in the SPME fiber, which led to a new calibration method, kinetic calibration.6,7 The kinetic calibration method is based on the desorption of the internal standards, which are preloaded in the extraction phase and then used to calibrate the extraction of the analytes. This method was also extended to solid-coated SPME, based on the isotropy of adsorption and desorption.8 To date, the kinetic calibration method has been successfully applied in environmental monitoring, food, and clinic drug analysis, in laboratory, and on-site analyses, as well as in vivo studies.8-14 Moreover, this method has been discussed in a number of academic reviews.15-17 Unfortunately, the reported kinetic calibration methods require the use of radioactive or deuterated compounds for the preloaded internal standards. The radioactive or deuterated substances are relatively expensive and the availability is limited. In addition, they may not be suitable for in vivo studies. Kinetic calibration has not been used for the in vivo sampling of tissues or plants.18 (4) Ai, J. Anal. Chem. 1997, 69, 1230-1236. (5) Ai, J. Anal. Chem. 1997, 69, 3260-3266. (6) Chen, Y. O’Reilly, J.; Wang Y.; Pawliszyn, J. Analyst 2004, 129, 702-703. (7) Chen, Y.; Pawliszyn, J. Anal. Chem. 2004, 76, 5807-5815. (8) Zhou, S. N.; Zhang, X.; Ouyang, G.; Es-haghi, A.; Pawliszyn, J. Anal. Chem. 2007, 79, 1221-1230. (9) Wang, Y.; O’Reilly, J.; Chen, Y.; Pawliszyn, J. J. Chromatogr., A 2005, 1072, 13-17. (10) Ouyang, G.; Zhao, W.; Pawliszyn, J. Anal. Chem. 2005, 77, 8122-8128. (11) Ouyang, G.; Pawliszyn, J. Anal. Chem. 2006, 78, 5783-5788. (12) Zhao, W.; Ouyang, G.; Alaee, M.; Pawliszyn, J. J. Chromatogr,, A 2006, 1124, 112-120. (13) Bragg, L.; Qin, Z.; Alaee, M.; Pawliszyn, J. J. Chromatogr. Sci. 2006, 44, 317-323. (14) Musteata, F. M.; Musteata, M. L.; Pawliszyn, J. Clin. Chem. 2006, 52, 708715.
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Figure 1. Schematic of the absorption and desorption processes between the liquid coating of SPME fiber and the tissue matrix. A linear concentration gradient is assumed in both the fiber coating and the tissue medium when the experimental conditions are constant. (A) is the absorption process and (B) is the desorption process. Cs is concentration of the analyte in the tissue matrix, C′s is the concentration of the analyte in the sample at the interface of the fiber coating and the tissue, Cf is the concentration of the analyte in the coating at the interface of the fiber coating and the tissue, C′f is the concentration of the analyte in the coating at the interface of the fiber coating and the stainless steel wire, and δf is the thickness of the fiber coating, r is the distance measured perpendicular to the area considered for an analyte, @ represents an analyte molecule with the arrow line indicating the diffusion route, and b is the solid tissue, which the analyte does not pass through.
Finally, the time constant required for kinetic calibration is previously obtained from extraction or desorption time profiles by a nonlinear approach, which is not suitable for an extraction with a longer equilibrium time such as tissue sampling. To overcome the limitations mentioned above, one novel calibration method using dominant pre-equilibrium desorption was developed for on-site sample preparation or soft tissue sampling and quantitative analysis. In this method, the target analytes serve as the internal standards; therefore, radioactivity or deutarated standards are not necessary. Concentration profiles were initially employed to validate isotropic behavior between absorption and desorption, which are more accurate and convenient than reported time profiles. It was found that dominant pre-equilibrium desorption not only offered a shorter sampling preparation time but also provided a linear approach to obtain time constants. This study is also the first report of the use of SPME for in vivo tissue sampling and quantitative analysis. THEORETICAL CONSIDERATIONS When a SPME fiber is exposed to a simple liquid medium (i.e., without cells), mass diffusion of an analyte from the sample matrix to the SPME polymer coating and from the surface of the fiber coating to its inner layers is considered to be the rate-determining step that slows down the process of reaching an equilibrium.19 This process follows Fick’s first law of diffusion:4,20
J≡
∂Cf ∂Cs 1 ∂n ) -Df ) -Ds A ∂t ∂x ∂x
(3)
where J is the mass flux of the analyte from the fiber to the sample matrix, A is the surface area of the fiber, ∂n is the amount of (15) Ouyang, G.; Pawliszyn, J. TrAC, Trends Anal. Chem. 2006, 25, 692-703. (16) Ouyang, G.; Pawliszyn, J. Anal. Bioanal. Chem. 2006, 386, 1059-1073. (17) Musteata, F. M.; Pawliszyn, J. TrAC, Trends Anal. Chem. 2007, 26, 36-45. (18) Lord, H. L.; Mo¨der, M.; Popp, P.; Pawliszyn, J. Analyst 2004, 129, 107108. (19) Louch, D.; Motlagh, S.; Pawliszyn, J. Anal. Chem. 1992, 64, 1187-1199. (20) Benveniste, H.; Hansen, A. J.; Ottosen, N. S. J. Neurochem. 1989, 52, 17411750.
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analyte that has absorbed to the fiber surface during time period ∂t, Df and Ds are the diffusion coefficients of the analyte in the fiber coating and sample matrix, respectively, and Cf and Cs are the concentrations of the analyte in the fiber and the sample matrix, respectively. The dynamic processes and isotropic behavior in a simple liquid matrix have been previously reported.4,7 In the case of a tissue (Figure 1) with a total volume V and interstitial space volume V0, the flux J to the fiber is20
J≡
∂Cf ∂Cs 1 ∂n ) -Df ) -RD hs A ∂t ∂x ∂x
(4)
where R is the effective volume fraction (V0/V) and Ds is the diffusion coefficient in the tissue. Flux of diffusion in a soft tissue is proportional to the R value, which is usually lower than 1. On the other hand, the diffusion path length in vivo is clearly greater than that existing in a free solution, because of the tortuosity (λ) of the route, which can be observed in Figure 1. Therefore, the diffusion coefficients in a free liquid medium (Ds) are usually higher than those found in a porous matrix such as a soft tissue. From eq 4, the same mathematical treatment of the absorption process is performed to generate4
n/ne ) 1 - exp(-at)
(5)
where n is the extracted amount of analyte on the surface of the fiber coating in moles after the exposure time t, ne is the extracted amount of the target analyte at equilibrium, and a is the time constant that is used to describe how quickly the equilibrium can be reached, which is defined by eq 6.
a)
2ADfRD h s(KVf + Vs) 2KVsVfDfr + VsVfRD h sδf
(6)
where r is the distance measured perpendicular to the area considered and δf is the thickness of the polymer coating.
Equation 6 illustrates that the time constant for a soft tissue is different from that of a simple liquid matrix.4 After a SPME liquid coating fiber is preloaded with an analyte, it is exposed to a tissue matrix and desorption of the analyte from the fiber occurs (Figure 1B). From eq 4, the same mathematical treatment for the process of desorption leads to eq 7,7,9
Q - qe ) exp(-at) q0 - qe
(7)
where Q is the remaining amount of the analyte in the fiber at time t, q0 is the preloaded amount of the analyte, qe is the remaining amount of analyte in the fiber at equilibrium, and a is the time constant. qe is considered not to be negligible when Kfs is large. Based on eqs 5 and 7, the sum of n/ne and (Q - qe)/(q0 - qe) should be 1 at any absorption/desorption time, which is expressed by,
Q - qe n + )1 ne q 0 - q e
(8)
Equation 8 indicates the possibility of kinetic calibration using SPME, as previously mentioned. However, qe may be difficult to obtain when a long equilibrium time exists for a living tissue sampling. In addition, wound effects could occur when a fiber placed for an extensive period in the living tissue. To solve this problem, eq 7 can be expressed as
Q ) [exp(- at)]q0 + [qe - qe exp(-at)]
(9)
From eq 9, a plot of Q versus q0 yields a straight line with a slope of exp(-at) and a y-intercept of [qe - qe exp(-at)] when the pre-equilibrium desorption time and experimental conditions are constant. Thus, the value of a can be calculated from the slope of the linear regression equation, and qe is obtained from the value of the intercept. In addition, eq 9 can be applied to both simple liquid media and semisolid matrixes. The value of the time constant can be used to obtain the initial concentration of the analyte in sample matrix by eqs 5 and 2. When the fiber is preloaded with a large amount of the analyte and then exposed to the sample matrix, desorption becomes dominant and extraction can be negligible. This dominant desorption can be employed to calibrate absorption, which is named as kinetic calibration using dominant pre-equilibrium desorption. It is suitable for on-site and in vivo sampling. EXPERIMENTAL SECTION Chemicals and Supplies. Carbofuran, Propoxur, carbaryl, aldicarb, and promecarb were supplied from Riedel-de Hae¨n, with greater than 99.5% purity. Acenaphthene, fluorene, anthracene, and pyrene were purchased from Supelco (Bellefonte, PA). Acetonitrile and methanol (HPLC grade) were obtained from Fisher Scientific (Nepean, ON, Canada). Helium was supplied by Praxair (Kitchener, ON, Canada) and was of ultrahigh purity. Stock standard solutions (1000 mg/L) of five pesticides were prepared in acetonitrile and stored at 0-4 °C in the dark. Working
solutions were prepared by a series of 10-fold successive dilution for quantitative calibration and sample preparation. The SPME fiber holder and 100-µm poly(dimethylsiloxane) (PDMS) fiber were obtained from Supelco (Oakville, ON, Canada) for the analysis of polycyclic aromatic hydrocarbons (PAHs). The 100-µm PDMS fiber was conditioned at 250 °C for 1 h prior to use. Self-made 165-µm PDMS fibers were used for both in vitro and in vivo pesticide sampling. PDMS hollow fiber membrane tubing (Helixmark, Carpinteria, CA) was cut into 1.0-cm portions. Each PDMS portion was placed over one stainless steel wire, the plunger of 10-µL syringe (Hamilton). Before PDMS coatings were mounted, and wires were sonicated in deionized water and acetone to ensure their cleanliness. The 165-µm PDMS fibers were conditioned in methanol for 1 h in static mode and then in Nanopure water for 1 h prior to use. For small-volume analysis, a polyethylene insert, which has a volume of 200 µL, was positioned in a 2-mL vial. The system for the generation of the standard PAH aqueous solution has been previously described.21 The temperature of the permeation chamber was maintained at 30 ( 1 °C with a temperature controller (Omegalux), to minimize the effect of the temperature on the flow-through system. The jade plants (Crassula ovata) were purchased from a local greenhouse. Instrumental Analysis. A Saturn 3800 GC/2000 ITMS system fitted with a SPB-5 column (30 m, 0.25-mm i.d., 0.25-µm film thickness) (Supelco, Mississauga, ON, Canada) was used for the analysis of the PAHs. The MS system was operated in the electron ionization mode and tuned with perfluorotributylamine. The carrier gas, helium, was set to a flow rate of 1 mL/min. The 1079 injector was set to 270 °C, for the SPME injections. For the liquid injections, it was set at 40 °C and then increased to 250 °C at a rate of 100 °C/min. The column temperature was maintained at 40 °C for 2 min and then increased by 30 °C/min increments to 250 °C, held for 5 min, and then increased by 30 °C/min increments to 280 °C and held for 15 min. The total run time was 30 min. A mass scan from 40 to 300 was obtained, and the base peak of each compound was selected and integrated. The instrument was checked on a daily basis by calibration with a liquid midpoint calibration standard. Any deviation in the area counts greater than 15% required reinjection of that standard; if the deviation was still greater than 15%, the instrument was recalibrated with a five-point calibration plot. Peak shape quality, resolution, and retention times were also carefully monitored to ensure that the chromatography was within the required specifications. Pesticide analyses were performed on an LC-MS/MS system consisting of a Shimadzu 10AVP liquid chromatograph with a system controller and dual binary pumps interfaced to a CTCPAL autosampler and an MDS Sciex API 3000 tandem mass spectrometer. The assays were carried out as described previously.8 In addition, the propoxur and aldicarb transitions were monitored from m/z 210.1/111.2 and 116.2/89.0, respectively. Absorption and Desorption. For the PAH analysis, a CTC CombiPal autosampler (Zwingen, Switzerland) using the associated Cycle Composer software (Version 1.4.0) was employed for the experiments with a 2-min preagitation period (at 500 rpm) (21) Ouyang, G.; Chen, Y.; Pawliszyn, J. J. Chromatogr., A 2006, 1105, 176179.
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and 500 rpm agitation at room temperature (25 °C) for absorption, analyte loading and desorption. Four experiments were designed to verify isotropism between absorption and desorption in the aqueous solutions. The first experiment was designed for absorption with a constant concentration and variable time, which produced absorption time profiles (n-t). The conditioned 100µm fiber was directly immersed in 10 mL of aqueous solutions with 0.100 µg/mL acenaphthene, fluorene, anthracene, and pyrene, for a specified period of time (5, 10, 15, 30, 45, 60, 90, 120, 180, or 240 min). The second experiment was designed for desorption with a constant concentration and variable time, which generated desorption time profiles (Q-t). The fiber was immersed in an aqueous solution containing 0.100 µg/mL PAHs in a vial for 1 h to preload the analytes. Subsequently, each preloaded fiber was exposed to 10 mL of PAH-free water in each vial to allow for desorption for a specified period of time (5, 10, 15, 30, 45, 60, 90, 120, 180, or 240 min). The third experiment was designed for absorption with variable concentrations and a constant preequilibrium extraction time, and absorption concentration profiles were obtained (n-ne). The conditioned fibers were directly exposed in the vials that contained 0.080, 0.040, 0.020, or 0.010 µg/mL PAHs for 20 (to obtain n) or 240 min (to obtain ne), to perform absorption. The last experiment was designed for desorption with variable concentration and a constant pre-equilibrium desorption time to make desorption concentration profiles (Qq0). The fiber was exposed to a 10-mL sample of aqueous solution that contained 0.080, 0.040, 0.020, or 0.010 µg/mL PAHs for 30 min, to allow for analyte preloading. Each fiber was then exposed to 10 mL of PAH-free water in each vial for 20 min to perform desorption (to get Q). To obtain q0, desorption was not performed in water. After absorption or desorption, the analytes remaining in the fiber were desorbed in the injector of the GC/MS for quantitative analysis. For the pesticide analysis, agarose gel (1% w/v) was spiked with various concentrations of pesticides for in vitro absorption and desorption experiments to verify isotropism and kinetic calibration. The gel was made by mixing 0.5 g of agarose (Agarose 15, BDH Chemicals Ltd., Poole, England) with 50 mL of phosphatebuffered saline, pH 7.4 (PBS), in a beaker. After the mixture was heated and became transparent and homogeneous, 1.9 mL was transferred to a 2-mL vial, which contained a 100 µL of aqueous PBS pesticide solution. The 2-mL vial was then capped and vortexed. Finally, the mixture was cooled to ambient temperature, yielding a semisolid gel. The agarose gels were used for further experiments after 2 or 3 h of stabilization. All of the experiments using the gel medium were performed under static mode. Four experiments were designed as mentioned above to verify isotropism between absorption and desorption. The first experiment was performed to produce absorption time profiles. The conditioned fiber was directly immersed in the vial containing the gel with 2.0 µg/mL carbofuran, propoxur, carbaryl, and aldicarb, for a specified period of time (2, 5, 10, 20, 30, 60, 90, 120, 150, 200, 250, or 300 min). The second experiment was performed to generate desorption time profiles. Each fiber was immersed in a PBS solution containing 2.0 µg/mL pesticides in a vial for 1 h to perform the analyte preloading. Then the preloaded fiber was exposed in a vial containing the pesticide-free agarose gel to allow for the desorption process, for a specified period of time (2, 5, 10, 20, 30, 60, 90, 120, 150, 200, 250, or 300 min). The third experiment was performed to develop an absorption concentration profile. The 484
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conditioned fiber was directly exposed to the agarose gel containing the pesticides (0.0625, 0.125, 0.25, or 0.50 µg/mL) for 20 (to obtain n) or 300 min (to obtain ne), for absorption. The last experiment was performed to develop desorption concentration profiles. The fiber was exposed in a vial containing the PBS (pH 7.4) pesticide aqueous solution (at concentrations of 0.25, 0.50, 1.00, or 2.00 µg/mL) for 1 h, for analyte preloading. The fiber was then exposed to a vial that contained pesticide-free agarose gel for 20 min (to obtain Q) for desorption. To obtain q0, desorption was not performed in the agarose gel. After the process of absorption or desorption above, the analytes remaining in the PDMS fiber were desorbed in a 200-µL insert containing 75 µL of acetonitrile for 1 h under static mode. Then, 75 µL of a 50 ng/mL promecarb aqueous solution, the internal standard for LC-MS/ MS, which is used to compensate for the variation of injection volume, was added to the desorption solution in the 200-µL insert. After the solution was vortexed, a 20-µL aliquot of the final solution was automatically injected into the LC-MS/MS for quantitative analysis. Dominant Pre-equilibrium Desorption. In the case of PAHs, the preloading was performed in an aqueous PAH solution (at a concentration of 10.0, 20.0, 40.0, or 80.0 ng/mL for 30 min at 500 rpm. Three types of aqueous solutions were evaluated for desorption. The first solution was PAH-free. The second solution contained PAHs with 4-fold lower concentrations (2.5, 5.0, 10.0, or 20.0 ng/mL, respectively) compared to the preloading solution. The third solution contained 8-fold lower concentrations (1.25, 2.5, 5.0, or 10.0ng/mL, respectively). The desorption was performed at 500 rpm for 20 min. For pesticide analysis, the preloading was performed in the agarose gel containing 0.25, 0.50, 1.00, or 2.00 µg/mL pesticide, for 20 min. Then the fibers with the preloaded analytes were exposed to the pesticide-free gel for 20 min, as well as the gel containing 0.0625, 0.125, 0.25, or 0.50 µg/mL pesticides, respectively, which were 4-fold lower compared to the concentration in the preloaded gel matrices. Distribution Constants (Kfs). For pesticide analysis, two methods were used to measure the distribution constants (Kfs) between the fiber coating and the sample matrix. The first method involved the use of jade leaf juice as the sample matrix. The leaves from a jade plant without any pesticide soil application were cut and ground with a mortar and pestle. The juice was filtered through 0.2-µm Acrodisc Syringe Filters (Ann Arbor, MI) to remove the fine particles. The leaf juice was then spiked with pesticides at a concentration of 0.25 µg/mL. The PDMS fiber was exposed to the juice matrix for extraction for 180 min at 500 rpm and 25 °C. The second method to obtain Kfs was to use the agarose gel (1%, w/v) made from PBS (pH 7.4) with a pesticide concentration of 0.25 µg/mL. The extraction was performed under static mode and 25 °C with an equilibrium time of 300 min. On-Site and in Vivo Application. The PAH concentrations in the flow-through system were determined by SPME direct extraction. The extraction was performed for 20 min in the sampling cylinder with a flow rate of ∼0.25 cm/min. The process was repeated three times using the same fiber. Then, dominant pre-equilibrium desorption was done to perform kinetic calibration. The preloading was performed in the aqueous solutions for 30 min at 500 rpm. The concentrations for fluorene, anthracene, and pyrene in the loading solutions were 10.0, 20.0, 40.0, or 80.0 ng/ mL. The concentration of acenaphthene in the loading solutions was 4-fold higher. The fiber was inserted in the sampling cylinder to perform dominant pre-equilibrium desorption for 1 h. This
Figure 2. Locations for SPME absorption and desorption as well as two sampling points (1 and 2).
process was repeated using the same fiber with the different preloading amounts. The jade plants with the pesticide soil application were employed for in vivo sampling. A total of 0.01 g of each pesticide (Chem Service, West Chester, PA) was weighed and mixed. The mixed fine particles were spilled on the surface of the soil. Then water was sprayed to allow the solid pesticides to gradually dissolve and diffuse to the soil. Around 15 mL of water was applied to each plant every other day. In vivo experiments were carried out at room temperature for technical convenience. The absorption fibers were conditioned without any analyte preloading before the in vivo extraction. The desorption fibers were preloaded with analytes by exposing the fibers to the vials containing the PBS (pH 7.4) aqueous solutions spiked with pesticides at concentrations of 1.0, 2.0, 4.0, and 8.0 µg/mL for 20 min. As soon as the holes were drawn with a 21-gauge hypodermic needle, the absorption fibers and the desorption fibers were simultaneously inserted into holes in two jade plant leaves for 20 min to perform absorption and desorption. Figure 2 illustrates the locations for SPME absorption and desorption as well as two sampling points (1 and 2). Four fibers for desorption were inserted in the same leaf within 1 cm. The analyte desorption from the fibers to acetonitrile and the preparations of solutions for LC-MS/MS analysis were the same as mentioned above. (Note: It was difficult to perform the experiments simultaneously. Each fiber was inserted in the sample matrix at intervals of 1 min.) RESULTS AND DISCUSSION Primary Considerations Prior to On-Site and in Vivo Study. The first step involved in developing a SPME method involves the choice of a suitable fiber. The PDMS fiber was applied to the flow-through system and on-site application due to its high affinity to PAHs. In addition, the PDMS coating was selected for pesticide sampling for a number of reasons. The PDMS coating is high-purity medical grade, i.e., biocompatible. Thus, the approach can be further applied to animal or human studies. In addition, as a liquid coating, PDMS does not have the disadvantage of competition or displacement effects compared with a solid coating. The fibers were made in-house, due to the consideration of size and cost of using the fibers. Generally speaking, com-
mercially available fibers are expensive and are not suitable for single-use and in vivo experiments. Internal standard loading onto an SPME fiber is an important step in order to obtain precise results.22 The preloading process in this study was performed in aqueous, PBS solutions or gel, instead of the sample headspace. This approach was used to wet the surface of the fiber with a layer of water prior to any absorption or desorption. This wetting procedure kept the surface of the fiber from drying, which could potentially influence the fiber performance. It is found that there is not a big difference using water solution, a PBS solution, or an agarose gel for pesticide preloading. The purpose of preloading is to allow a certain amount of analytes to go to the fiber coating. It is fine as long as the same preloading medium is used for the same batch experiment. The headspace preloading approach causes the fiber coating surface to dry (or air bubble on the fiber surface), which results in poor reproducibility. Also, although pesticides can be preloaded to the fiber using a headspace approach under a higher temperature (higher than room temperature), it makes experimental procedures too complicated. An agarose gel spiked with pesticides was chosen as a mimic system for an in vitro study based on the following issues. At a certain concentration, agarose gel prevents thermal convection but does not obstruct free diffusion significantly.23 Although an agarose gel is different from a living tissue, in vitro experiments can be very useful to find mass-transfer phenomena in both SPME coatings and a semisolid matrixes. It may also yield relevant information for in vivo study. Verification of Isotropism between Absorption and Desorption. The kinetics of absorption and desorption were performed in both the aqueous matrix and the agarose gel medium to validate that the desorption of a standard from the extraction phase into the sampling matrix was isotropic to the absorption of the analyte into the extraction phase from the sample matrix. Time profiles were initially used for this verification. The concentrations of analytes (PAHs or pesticides) in the sample matrix (aqueous solution or gel) remained the same in spite of different absorption times. In addition, the preloaded amounts of analytes were kept equal as the desorption time was changing. This approach has not been previously used for a semisolid medium although it has been performed in the aqueous solutions.7,8,12,13 The sum of n/ne and (Q - qe)/(q0 - qe) was close to 1 for all of the PAHs and pesticides used in this study, which is consistent with eq 8. One example is shown in Figure 3. To further evaluate the isotropism, the time constants from absorption were compared with those from desorption. Rearrangement of eqs 5 and 7 leads to eqs 10 and 11, respectively,
( )
(10)
Q - qe ) - at q0 - qe
(11)
n ln 1 ) - at ne
(
ln
)
The linearized absorption and desorption time constant profiles can be obtained with ln(1 - n/ne) or ln[(Q - qe)/(q0 - qe)] as (22) Zhao, W.; Ouyang, G.; Pawliszyn, J. Analyst 2007, 132, 256-261. (23) Nicholson, C.; Phillips, J. M. J. Physiol. 1981, 321, 225-257.
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10 and 11 accurately describe the kinetics of SPME desorption and absorption in both an aqueous and gel medium. In addition, the isotropic processes were observed using the 100-µm PDMS fiber in the agarose gel medium. The data are not shown here. Concentration profiles were used to verify the isotropic behavior between absorption and desorption as well. This approach is reported for the first time. The absorption or desorption times remained the same in spite of the diverse analyte concentrations in the aqueous solution or gel for absorption or the different preloaded amounts in the fiber for desorption. Equation 5 is then rearranged to eq 12,
n ) [1 - exp(-at)]ne Figure 3. Propoxur absorption (0) and desorption (]) time profiles. The absorption was performed in agarose gel (1%, w/v) containing 2 µg/mL carbofuran, propoxur, carbaryl, and aldicarb in static mode and 25 °C. For desorption profile, the analytes were preloaded in a PBS (pH 7.4) aqueous solution containing 2.0 µg/mL pesticides for 1 h, and then the fiber was desorpiton in a pesticide-free gel. The 165-µm PDMS fibers were used to produce the profiles. ∆, the sum of n/ne, and (Q - qe)/(q0 - qe). Table 1. Time Constants a for Absorption and Desorption of PAHs and Pesticides from Time Constant Profilesa time constant a (min-1) (R2)
acenaphthylene fluorine anthracene pyrene carbofuran propoxur carbaryl aldicarb
absorption
desorption
0.0253 ( 0.0022(0.990) 0.0228 ( 0.0024 (0.996) 0.0147 ( 0.0018 (0.992) 0.0092 ( 0.0014 (0.990) 0.0130 ( 0.0010(0.990) 0.0142 ( 0.0016 (0.994) 0.0126 ( 0.0010 (0.994) 0.0131 ( 0.0012 (0.991)
0.0252 ( 0.0024 (0.993) 0.0217 ( 0.0030 (0.990) 0.0153 ( 0.0026 (0.984) 0.0099 ( 0.0012 (0.990) 0.0126 ( 0.0021 (0.987) 0.0136 ( 0.0020 (0.991) 0.0127 ( 0.0008 (0.991) 0.0129 ( 0.0016 (0.991)
a Ten and 11 data points were used to calculate each time constant for PAHs and pesticides, respectively.
the y-axis, and t as the x-axis, where the regression slope is -a. Table 1 lists the time constants of absorption and desorption for the different analytes. Time constants for the same compound remained similar between absorption and desorption with less than 5% relative standard deviation (RSD) observed in most cases. It also suggested the isotropic behavior of absorption and desorption. Figure 4 illustrates one example, which demonstrates that eqs
(12)
From eq 12, a plot of n versus ne yields a straight line with a slope of [1 - exp(-at)] when the absorption conditions and sampling time are fixed. It can also be applied to eq 9 with a slope of exp(-at). The sum of the slopes from the absorption and desorption concentration profiles should be 1 for a fixed preequilibrium time. Figure 5 shows one example of absorption and desorption concentration profiles. In addition, Table 2 summarizes the slopes of aborption and desorption lines, as well as the sum of the slopes from two lines, which is very close to 1. It suggests that an isotropic process exists between absorption and desorption. Table 2 lists the time constants calculated from the slopes as well. It is evident that the values of the time constants from the absorption and desorption are similar. Time constant, a, which is defined in eq 6, is a measure of how quick an absorption or desorption can be reached in a semisolid medium. It is affected by several factors including the properties and dimension of the fiber coating, the structure and properties of the analyte, and components of the aqueous or tissue matrix. For a living tissue, the effective volume fraction, R, and the diffusion coefficient, D h s, make the value of the time constant in a soft tissue different from that of a simple liquid matrix or other tissues. The tortuosity factor (λ2 ) D h s/Ds) explains the increased diffusion pathway that occurs in vivo due to the presence of impermeable materials.24 There is not a significant difference of the time constants for the four pesticides studied, based on the results presented in Tables 1 and 2. This is likely caused by the large pores that are present in agarose gel.25 Comparing Table 1 with Table 2, the values of time constants for the same compound are close for both the absorption and desorption. This suggests that both time profiles and concentration
Figure 4. Carbofuran absorption (0) and desorption (]) time constant profiles. The same experimental conditions were set as in Figure 3. 486
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Table 2. Slopes for Aborption and Desorption from Concentration Profiles, the Sum, and Time Constants Calculated from Slopes time constant, a, (min-1)b
slopea
acenaphthylene fluorine anthracene pyrene Carbofuran Propoxur carbaryl aldicarb
absorption 1-exp(-at) (R2)
desorption 1-exp(-at) (R2)
sum of slopes
absorption
desorption
0.4019 ( 0.0173 (0.995) 0.3622 ( 0.0177 (0.980) 0.2532 ( 0.0144 (0.993) 0.1577 ( 0.0054 (0.991) 0.2188 ( 0.0090 (0.994) 0.2350 ( 0.0089 (0.987) 0.2225 ( 0.0042 (0.988) 0.2255 ( 0.0120 (0.995)
0.6166 ( 0.0154 (0.999) 0.6526 ( 0.0183 (0.997) 0.7252 ( 0.0377 (0.997) 0.8420 ( 0.0539 (0.998) 0.7675 ( 0.0215 (0.996) 0.7508 ( 0.0233 (0.997) 0.8037 ( 0.0193 (0.996) 0.7605 ( 0.0274 (0.993)
1.0185 1.0148 0.9784 0.9997 0.9863 0.9858 1.0262 0.9860
0.0257 ( 0.0014 0.0225 ( 0.0014 0.0146 ( 0.0010 0.0086 ( 0.0003 0.0123 ( 0.0006 0.0134 ( 0.0006 0.0126 ( 0.0003 0.0128 ( 0.0008
0.0242 ( 0.0013 0.0213 ( 0.0014 0.0161 ( 0.0026 0.0086 ( 0.0032 0.0132 ( 0.0014 0.0143 ( 0.0016 0.0109 ( 0.0012 0.0137 ( 0.0018
a Four data points were used to obtain each slope. b Time constant uncertainty were calculated using propagation of uncertainty from those of slopes.
Figure 5. Carbaryl absorption (]) and desorption (∆) concentration profiles. The absorption was performed by the exposure of the fiber to the agarose gel (1%, w/v) containing 0.0625, 0.125, 0.25, or 0.50 µg/mL pesticides for 20 (to obtain n) or 300 min (to obtain ne). For the desorption profile, the fiber was exposed to the PBS (pH 7.4) aqueous solution containing 0.25, 0.50, 1.00, or 2.00 µg/mL pesticides for 1 h under static mode for analyte preloading, and then the analytes was desorbed from the fiber in the pesticide-free agarose gel for 20 min under static mode (to obtain Q). To obtain q0, desorption was not performed in the agarose gel.
profiles could be employed for kinetic calibration. However, there are three advantages associated with concentration profiles. First, the use of concentration profiles provides a shorter sample preparation time. It is typically suitable for on-site analysis, and especially for in vivo sampling. A long equilibrium extraction may be impractical because the concentrations of the analytes in the living system could change within a short period of time. A potential wound effect is another concern. Second, it is more robust and easier to obtain time constants using concentration profiles. When an experiment for a time profile is performed, little variation in the value, n/ne or (Q - qe)/(q0 - qe), close to the equilibrium point can cause a significant change in the time constant due to the relationship of the natural logarithm. Finally, compared to the use of time profiles, concentration profiles are more accurate with lower RSD and smaller error bars, as observed in Tables 1 and 2, as well as Figures 3-6. Based on these observations, concentration profiles were further investigated in this study. There are a number of steps to determine the pre-equilibrium absorption and desorption time to produce concentration profiles.
The first step is to find the equilibrium time by extraction experiments or absorption time profiles. Then, the pre-equilibrium time for concentration profiles can be set at 1/10 of the equilibrium time. This time point is the crossing point of absorption and desorption time profiles that can also be explained by the mathematical treatment of eqs 5 and 7. Around half of the amount equilibrium extraction from the sample matrix as well as a certain amount of desorption from the preloading fiber can be observed at this time point, as illustrated in Figure 3. In addition, the fiber thickness and the coating material can be chosen to obtain an ideal pre-equilibrium desorption time or the crossing time point. Dominant Pre-equilibrium Desorption. Dominant preequilibrium desorption was evaluated to determine whether desorption is dominant and extraction is negligible when the preloaded amount of an analyte in the fiber is larger than the potential extracted amount from the sample matrix. The experiment was performed to determine if the desorption rate could be influenced by small amounts of target analytes in the sample matrix. It was found that the desorption rate was constant when the preloaded amounts of the analyte in the desorption fiber were 4-fold higher than the potential extracted amounts from the sample matrix, as shown in Figure 6. Two separate desorption lines result in similar slopes, exp(-at), for aldicarb. Since the preloaded internal standards are the same as the extracted analytes, a certain distance should be kept between the fibers for desorption and extraction to avoid diffusion of the desorbed analytes to the fibers for extraction when an experiment is designed. Application to Real Sample Matrix. To test the kinetic calibration for on-site and in vivo tissue sampling, this method was used to quantify PAHs in the flow-through system with GC/ MS and the pesticides in the leaves of jade plants with LC-MS/ MS, respectively. 1. Distribution Constants (Kfs). Since the kinetic calibration belongs to an equilibrium standardization technique, the distribution constants should be known in order to estimate the initial concentrations of analytes in a sample matrix. Distribution constants of PAHs have been previously reported.26 In the case of pesticides, the purpose of utilizing agarose gel (1%, w/v) made from PBS (pH 7.4) as the sample matrix was to prove the reported (24) Safford, R. E.; Bassingthwaighte, J. B. J. Biophys. 1977, 20, 113-136 (25) Mikkelsen, S. R., Corto´n, E. Eds. Bioanalytical Chemistry; Wiley-Interscience: Hoboken, NJ, 2004. (26) Ouyang, G.; Cai, J.; Zhang, X.; Li, H.; Pawliszyn, J., submitted.
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Table 4. Slopes of Desorption Concentration Profiles Obtained from the Flow-Through System, Time Constants, a, Calculated PAH Concentrations, Limits of Detection, and Limits of Quantitation
compound acenaphthylene fluorine anthracene pyrene a
Figure 6. Comparison of two desorptions for aldicarb in the agarose gels (1%, w/v). The analyte preloading to the fiber was performed in the gels containing four pesticides of 0.25, 0.50, 1.00, or 2.00 µg/mL for 20 min. The analyte desorption from the fiber was performed for 20 min in the pesticide-free gel (∆) or the gel with 4-fold lower pesticide concentrations, i.e., 0.0625, 0.125, 0.25, or 0.50 µg/mL (]), compared to the preloading concentrations. Table 3. Distribution Constants (Kfs) of Pesticides and LC Retention Times (tr) (n ) 3)
carbofuran propoxur carbaryl aldicarb
Kfsa
Kfsb
tr (min)
73.2 ( 4.4 57.3 ( 3.1 83.9 ( 7.0 40.0 ( 1.7
81.7 ( 5.4 65.5 ( 4.8 80.6 ( 6.9 42.0 ( 2.4
2.80 2.76 3.05 2.34
a The sample matrix was the agarose gel (1%, w/v) made from PBS (pH 7.4). b The sample matrix was the filtered juice from the jade leaves.
assumption. It was assumed that extraction is essentially from extracellular fluid, which would have properties similar to the PBS buffer.18 The distribution constant for the direct extraction mode can be calculated from the following dependence obtained from eq 13,2
Kfs )
neVs Vf(C0Vs - ne)
(13)
Table 3 lists the distribution constants obtained from both approaches and LC retention times for liquid chromatograms. It was found that the values of Kfs from both approaches were similar, which proved the correction of the previous assumption. In addition, the values of the distribution constants matched those of LC retention times. Both the PDMS coating and C18 particles in the LC column are nonpolar. The higher hydrophobic property indicates that the compound has the higher value of distribution constant as well as the longer retention time. 2. Kinetic Calibration for PAHs and Pesticide Sampling. For the on-site sampling of PAHs, the flow-through system was used. Table 4 presents the slopes of the desorption concentration curves, the values of the time constants, and the measured PAH concentrations in the flow-through system. Excellent linearity was observed using dominant pre-equilibrium desorption with analytes as the internal standards. For pesticide sampling, although the isotropism was validated in a gel medium, the geometry and properties of a constructed 488 Analytical Chemistry, Vol. 80, No. 2, January 15, 2008
slope of desorptiona a exp(-at) (R2) (min-1) 0.9393 (0.999) 0.9067 (0.996) 0.8253 (0.998) 0.8493 (0.999)
concnb (µg/L)
0.00104 28.54 ( 3.08 0.00163 41.45 ( 1.87 0.00320 1.45 ( 0.11 0.00272 1.92 ( 0.16
LOD LOQ (µg/L) (µg/L) 6.64 6.91 0.31 0.22
22.13 23.02 1.03 0.73
Four data points were used to obtain each slope. b n ) 3.
gel medium could not be the same as those of a real tissue. Passage of the substance in vivo has to be expected to be different from in vitro.27 It is clear that in vitro calibration has scant application in SPME in vivo. To perform in vivo calibration, several factors had to be considered. The first factor is the homogeneity of the soft tissue. Since the diameter of the SPME fibers used in this study is by far larger than that of the cells in the living tissue, it can be assumed that the structure of the sampled tissue is homogeneous. The second factor is the number of desorption fibers that are used to construct a desorption concentration profile. A high affinity fiber to the target analyte is preferred in order to improve sensitivity, which could lead to a non-negligible qe. From eq 9, it is noted that the intercept is not equal to zero. Therefore, one-point calibration is not suitable for this case. To achieve more accurate results, four desorption points were applied for in vivo tissue sampling in this study. The third factor is the sampling positions, which should not allow the analytes desorbed from the desorption fibers to enter the absorption fiber. An ideal design is shown in Figure 2, where a neighboring leaf was used for the purpose of desorption to correct the matrix effects from sampling the original jade leaf. The basic assumption is that the neighboring leaf had the same structure and properties. A similar design could be applied to an animal experiment. If the sampling point is in the left leg or brain of a rat, the right one may be employed for desorpion. The fourth factor is the distance and orientation of the desorption fibers, which should not allow analytes desorbed from one desorption fiber to disturb desorption from other fibers. As shown in Figure 2, the fiber with lowest preloaded amount was close to the petiole or the stem, and the fiber with the highest amount was near the tip of the leaf. The transfer of systemic pesticides is in one direction from the sink to the shoot system, and the desorption fibers were kept a certain distance in between, so disturbance between desorption should not occur. It is also conformed with the excellent linearity obtained from the desorption concentration curves, which are shown in Table 5. Table 5 also presents the time constant values obtained from in vivo desorption concentration profiles. Also, one example is illustrated in Figure 7. Excellent linearity (R2 g 0.990) was observed for all pesticides tested in the living leaves of the jade plant. The values of the time constants show a difference among the four compounds tested, which indicates that the matrix effects compared to in vitro data in Table 1 and Table 2. Limits of (27) Scheller, D.; Kolb, J. J. Neurosci. Methods 1991, 40, 31-38.
Table 5. Slopes Obtained from in Vivo Desorption Concentration Profiles.a Time Constants, a, Limits of Detection, and Limits of Quantitation for in Vivo Analysis
compound
slope of desorption exp(-at) (R2)
a (min-1)
LOD (µg/L)
LOQ (µg/L)
carbofuran propoxur carbaryl aldicarb
0.8360 (0.990) 0.9353 (0.994) 0.9903 (0.999) 0.6633 (0.999)
0.00896 0.00334 0.00049 0.02053
5.33 11.25 12.67 4.23
10.74 15.67 44.11 11.26
a
Four data points were used to obtain each slope.
Figure 7. Carbofuran in vivo concentration desorption profile. The fiber was preloaded with carbofuran, propoxur, carbaryl, and aldicarb by exposing to the PBS (pH 7.4) aqueous solutions containing 1.0, 2.0, 4.0, or 8.0 µg/mL pesticides for 20 min under static mode. Then four fibers were simultaneously inserted to the same leaf of the jade plant within 1 cm to perform desorption for 20 min at 25 °C. It is illustrated in Figure 2.
detection (LODs) and limits of quantitation (LOQs) were obtained using 10 fibers to perform extraction in the leaves of a jade plant without any pesticide application (Table 5). LOD was calculated as a blank plus three times the standard deviation of the blank, and LOQ was equal to 10 times the standard deviation of repetitive measurements on a blank.28 LODs and LOQs of PAHs are listed in Table 4.
It was found that the pesticide was not homogeneously distributed in the leaf. The concentration of the pesticide at the sampling point 1 was higher than that of point 2 (Figure 2). These results indicate that these pesticides are xylem mobile. When a fiber was inserted to a leaf, the whole leaf could be considered as the same environment except pesticides. However, compared to the large amount of pesticides in the fibers, the small amount of pesticides presented in the leaf did not influence the dominant desorption of analytes from the fiber. This is the reason to employ the approach of dominant pre-equilibrium desorption. Sampling point 1 was employed to get concentration-changing profiles (Figure 8) obtained from a jade plant leaf after the systemic insecticides were applied directly to the growing medium, the soil. Systemic pesticides applied to the growing medium and taken up by plant roots have provided more than 50 days of residual activity, which matches the literature report with up to 12 weeks.29 It was noticed that a lower concentration of carbofuran was detected in the leaf compared to propoxur and carbaryl. It indicated that propoxur and carbaryl could be absorbed more by the jade plant than carbofuran, or carbofuran might bind strongly to various constituents of the soil. The absorption amounts and rate of pesticides from the soil to the leaves are influenced by several factors. The soil constituents, moisture, and temperature can affect the diffusion of pesticides from the soil to the roots. Pesticide properties such as hydrophobicity also affect the entrance of pesticides into the root system. In addition, the pesticide transport in a plant is influenced by atmosphere humidity, light intensity, and so on. Aldicarb was not shown in Figure 8 because the experimental results for aldicarb were different from the others. High amounts of each pesticide (0.2 g), including aldicarb, were applied to the soil of one jade plant. After 40 days, a small but detectable aldicarb peak was found. Although aldicarb is a soilapplied systemic pesticide like propoxur, carbaryl, and carbofuran, it is the least favored uptake by the jade plant. This might be caused by selective uptake effects among the four systemic pesticides that were used. In addition, aldicarb is the least lipophilic one of the four tested, so it may not be as easy as others to partition into the hydrophobic cutin. The error bars were not put in Figures 7 and 8 for the following reasons. Since it was not
Figure 8. Concentration-changing profiles of carbofuran (×), propoxur (]), and carbaryl (2). It was obtained from a jade plant leaf in sampling point 1 (Figure 2). The extractions were performed at 25 °C for 20 min. The 165-µm PDMS fibers were used for extraction.
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possible to perform multiple experiments in a single leaf within 1 day due to the small or limited size of a leaf, a single experiment was done to obtain Figures 7 and 8 at intervals of 5 days. Although different levels of leaves (older and newly grown) were used for sampling, the concentration of a pesticide in different levels was different. Compared to lower level leaves (older), the upper level leaves (newly grown) contained pesticides with lower concentrations. This indicates that the concentration in the leaf reflected the procedure of time accumulation. The lower level leaves grew first, so they tended to accumulate more pesticides. A fully mathematical description of the pesticide uptake by a jade plant is beyond the scope of this work. In vivo SPME sampling offers several advantages. It provides continuous sampling with minimal perturbation to the system under study. Fewer plants are used, and the method is a less invasive way than sampling in vitro or with other techniques. SPME is simpler and more convenient for on-site sampling or field monitoring than microdialysis, a commonly used in vivo technique, which requires a cumbersome syringe or osmotic pump during sampling. Although an osmotic pump is smaller and does not require a power supply, the flow rate delivered by an osmotic pump is less stable than that from a syringe pump.30 CONCLUSION This study introduces an accurate and reliable kinetic calibration method using dominant pre-equilibrium desorption for onsite and in vivo application by solid-phase microextraction. A newly proposed approach using the concentration profile with the preequilibrium approach not only validates isotropic behavior between absorption and desorption but also provides a linear approach to obtain a time constant, which is more accurate and convenient (28) Skoog, D. A., Holler, F. J., Nieman T. A., Eds. Principles of Instrumental Analysis; Saunders College Publishing: Philadelphia, PA, 1998. (29) http://www.pesticidesafety.uiuc.edu/newsletter/html/200204d.html. (30) Cooper, J. D.; Heppert, K. E.; Davies, M. I.; Lunte, S. M. J. Neurosci. Methods 2007, 160, 269-275.
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than that obtained from a time profile. Previously, kinetic calibration has been used with radioactive or deuterated internal standards that are expensive or sometimes not available. In the present work, solid-phase microextraction employs the target analytes as the internal standards by dominant desorption, which allowed desorption to calibrate absorption. Dominant pre-equilibrium desorption not only offers a shorter sample preparation time but also provides time constants for the purpose of quantitative analysis. This kinetic calibration method was successfully applied to on-site PAH sampling in a flow-through system and in vivo pesticide sampling in a jade plant (C. ovata). This developed SPME method is typically suitable for in vivo sampling in a field monitoring compared to microdialysis that requires a bulky syringe or osmetic pump. This method with modification should be applied to in vivo sampling in other biological organ systems. The proposed technique might also be useful for pollutant sampling in field monitoring, such as in a river, as long as the desorbed analytes from the desorption fibers do not diffuse to the absorption fibers. ACKNOWLEDGMENT This work has been financially supported by the Natural Science and Engineering Research Council of Canada. We acknowledge the assistance of Dr. Peterson and her research group as well as Lynn Hoyles from the department of Biology, University of Waterloo. The authors also thank their fellow research group members for useful discussions. NOTE ADDED AFTER ASAP PUBLICATION This article was posted ASAP on December 20, 2007. Two equations in text were clarified by addition of parentheses, and the paper was reposted on December 26, 2007. Received for review September 5, 2007. Accepted October 19, 2007. AC701871Q
