Anal. Chem. 1999, 71, 2146-2151
Artificial Receptor-Facilitated Solid-Phase Microextraction of Barbiturates Shu Li, Lifang Sun, Yongsoon Chung, and Stephen G. Weber*
Department of Chemistry, University of Pittsburgh, Pittsburgh, Pennsylvania 15260
A receptor for barbiturates, N,N′-Bis-[6-(2-ethylhexanoylamino)-pyridin-2-yl]-isophthalamide, was designed to dissolve in plasticizers of poly(vinyl chloride) (PVC). Microextractions using receptor-doped films of PVC were carried out as a function of receptor concentration. The effect of the concentration of the receptor on extraction yield is considerable for barbiturates that have significant binding to the receptor but negligible for very similar molecules that do not bind to the receptor strongly. Thus, it is the receptor’s ability in molecular recognition, not its generic ability as an H-bonding cosolvent, that is important. On the other hand, NMR data show that the receptor self-associates. A simple, approximate analysis is given to extract the amount of active receptor from the data. Receptor-enhanced extractions of barbiturates from urine are compared to extractions using a phosphate ester as solvent. In the quest for more information from increasingly complex samples, such as those generated by library or mixture synthesis,1 those from the environment,2 or from biological samples,3 enormous attention has been paid to increasing the resolution of separations and to increasing the sensitivity and specificity of detectors. At the same time very little attention has been paid to improvements in sample preparation. Microextraction is an extraction method in which both the quantity of consumed sample and produced extract are small. It is especially designed for subsequent analysis by methods such as gas chromatography,4 and capillary electrophoresis5 that have low mass detection limits. We report the first microextraction carried out with an artificial receptor. Furthermore, the extraction procedure is “green” as it uses no volatile organic solvents. If an extraction medium is selective, the extraction medium should at equilibrium contain relatively more of the analyte than (1) For example, recent papers are: Booth, R. J.; Hodges, J. C. J. Am. Chem. Soc. 1997, 119, 4882-4886; Marx, M. A.; Grillot, A.-L.; Louer, C. T.; Beaver, K. A.; Bartlett, P. A. J. Am. Chem. Soc. 1997, 119, 6153-6167, Flynn, D. J.; Crich, J. Z.; Devraj, R. V.; Hockerman, S. L.; Parlow, J. J.; South, M. S.; Woodard, S. J. Am. Chem. Soc. 1997, 119, 4874-4881; Studer, A.; Hadida, S.; Ferrito, R.; Kim, S.-Y.; Jeger, P.; Wipf, P.; Curran, D. P. Science 1997, 275, 823-826; Dunayevskiy, Y. M.; Vouros, P.; Wintner, E. A.; Shipps, G. W.; Carell, T.; Rebek, J. Proc. Natl. Acad. Sci. U.S.A. 1996, 93, 6152-6157. (2) Clement, R. E.; Yang, P. W.; Koester, C. J. Anal. Chem. 1997, 69, 251R287R. (3) Anderson, D. J.; Guo, B.; Xu, Y.; Ng, L. M.; Kricka, L. J.; Skogerboe, K. J.; Hage, D. S.; Schoeff, L.; Wang, J.; Sokoll, L. J.; Chan, D. W.; Ward, K. M.; Davis, K. A. Anal. Chem. 1997, 69, 165R-229R. (4) Pawliszyn, J.; Liu, S. Anal. Chem. 1987, 59, 1475-1478. (5) Li, S.; Weber, S. G. Anal. Chem. 1997, 69, 1217-1222.
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of other chemical species in the sample.6 One way to achieve selectivity is to use biological elements, such as antibodies,7 in extractions. Membranes8 and solid-phase materials9 with immunoaffinity have been applied successfully to the analysis of complex matrixes, such as environmental10,11 or biological fluids.12 The advantage of using antibodies is that high binding constants with selectivity to the target molecules can probably be achieved. Another route to selectivity is the use of synthetic receptors. In contrast to antibodies, synthetic receptors are small and simple molecules. The advantage of using synthetic receptors is that they are usually robust and predictable because of their simple structure. They may also be available in large quantities for low cost. Artificial receptors that gain their specificity and binding strength from hydrophobic effects work well in water, whereas those that function based on hydrogen bonding work well in the organic phase. Here we report on an approach with a synthetic receptor13-15 for the extraction of barbiturates into organic phases. To avoid saturation of the receptor population and to increase the extraction efficiency, the concentration of receptor sites in the extraction medium needs to be high. On the other hand, to achieve selectivity in an extraction requires the use of poor solvents. By using poor solvents, the extraction of unwanted species by virtue of their solubility in the solvent is minimized. Thus, the general problem exists to develop receptors that can function in, or for that matter even dissolve in, very poor solvents. Receptor 1a16 N,N′-bis-[6-(butyrylamino)-pyridin-2-yl]-isophthalamide (Figure 1) is effective in chloroform17 but fails in plasticized PVC because it is not very soluble.18 In the current work, we use solid-phase microextraction (SPME)19,20 using plasticized poly(6) Giddings, J. C. Unified Separation Science; John Wiley & Son, Inc.; New York, 1991; p 8. (7) Hennion, M.-C.; Barcelo, D. Anal. Chim. Acta 1998, 362, 3-34. (8) Dombrowski, T. R.; Wilson, G. S.; Thurman, E. M. Anal. Chem. 1998, 70, 1969-1978. (9) Pichon, V.; Rogniaux, H.; Fischer-Durand, N.; Rejeb, S. B.; Le Goffic, F.; Hennion, M.-C. Chromatographia 1997, 45, 289-295. (10) Ferrer, I.; Hennion, M.-C.; Barcelo´, D. Anal. Chem. 1997, 69, 4508-4514. (11) Pichon, V.; Chen, L.; Hennion, M.-C.; Daniel, R.; Martel, A.; Le Goffic, F.; Abian, J.; Barcelo, D. Anal. Chem. 1995, 67, 2451-2460. (12) Hennion, M.-C.; Cai, J. Y. Anal. Chem. 1996, 68, 72-78. (13) Bu ¨ hlmann, P.; Badertscher, M.; Simon, W. Tetrahedron 1993, 49, 595599. (14) Linton, B.; Hamilton, A. D. Chem. Rev. 1997, 97, 1669-1680. (15) Rebek, J. J. J. Mol. Recognit. 1992, 5, 83-88. (16) Chang, S. K.; Hamilton, A. D. J. Am. Chem. Soc. 1988, 110, 1318-1319 . (17) Valenta, J. N.; Dixon, R. P.; Hamilton, A. D.; Weber, S. G. Anal. Chem. 1994, 66, 2397-2403. (18) Valenta, J. N.; Weber, S. G. J. Chromatogr. 1996, 722, 47-57. (19) Zhang, Z.; Yang, M. J.; Pawliszyn, J. Anal. Chem. 1994, 66, 844A-853A. (20) Eisert, R. and Pawliszyn, J. Crit. Rev. Anal. Chem. 1997, 27, 103-135. 10.1021/ac980587o CCC: $18.00
© 1999 American Chemical Society Published on Web 04/21/1999
Figure 1. Receptors and barbiturates. Receptor 1a: R ) n-propyl, receptor 1b: R ) 3-heptyl, receptor 1c, 1d: macrocyclic receptors. Mephobarbital: X ) O,Y ) CH3; thiopental: X ) S,Y ) H; Other barbiturates: X ) O, Y ) H.
(vinyl chloride) (PVC) as the nonvolatile, reuseable extraction medium. We demonstrate that a receptor can be compatible with plasticizers that are poor solvents. Furthermore, the H-bondingbased molecular receptor acts as a specific receptor rather than as a polar cosolvent. Self-association of the receptor in the poor solvent occurs and decreases its efficacy. EXPERIMENTAL SECTION All of the chemicals, unless specified otherwise, were obtained from Aldrich (Milwaukee, WI) or Sigma (St. Louis, MO). Chloroparaffin was obtained from Fluka (Ronkonkoma, NY). Santicizer 141 is a gift from Monsanto (St. Louis, MO). Preparation of 1b, N,N′-bis-[6-(2-ethylhexanoylamino)-pyridin2-yl]-isophthalamide (Figure 1), adapted from Hamilton’s procedure,21 is as follows. The diamine resulting from condensation of 2 equiv of 2,6-diaminopyridine and isophthaloyldichloride was incubated for 8 h at room temperature with racemic 2-ethylhexanoyl chloride. The crude product was purified by chromatography on silica gel using 5% THF in CH2Cl2 as the eluent. After solvent evaporation, 1b was obtained as a pale yellow solid, mp 50-52 °C. 1H NMR (17.5 mM in CDCl3) δ 8.59 (2H, s, isophth-CONH), 8.49 (1H, br s, isophth-2H), 8.12 (2H, d, pyr-3H), 8.06 (2H, d, isophth-4,6H), 8.04 (2H, d, pyr-5H), 7.88 (2H, s, CH2CONH), 7.78 (2H, t, pyr-4H), 7.64 (1H, t, isophth-5H), 2.19 (2H, m, COCH), 1.73 (4H, m, COCH(CH2CH3)CH2), 1.57 (4H, m, COCH(CH2CH3)CH2CH2), 1.33 (4H, m, COCH(CH2CH3)CH2 CH2CH2), 1.33 (4H, m, COCHCH2CH3), 0.98 (6H, t, COCHCH2CH3), 0.93 (6H, t, (21) Chang, S. K.; Engen, D. V.; Fan, E.; Hamilton, A. D. J. Am. Chem. Soc. 1991, 113, 7640-7645 .
COCH(CH2CH3)CH2CH2CH2CH3). The molecular weight is 600 as measured by FAB-MS. The NMR titration data are obtained by titrating 0.5 mL of receptor CDCl3 solution (40% w/w) with CDCl3. The chemical shifts of the two pairs of amide NH protons of the receptor were monitored as the concentration changed. The association constant for 1b and phenobarbital in CHCl3 was determined by UV spectrophotometry according to Connors.22 Standard solutions were prepared by adjusting an aqueous solution of 10 barbiturates with 20 mM phosphate to pH 7.5. Spiked urine was prepared by mixing the standard solution of barbiturates with urine (1:9 or 1:49 v/v). The pH of the spiked urine was adjusted with sodium acetate and acetic acid to pH 5.5. Spiked serum was prepared by injecting into a vial containing lyophilized bovine serum, 25 µL of anhydrous acetic acid and 1 mL of the barbiturate standard solution, and bringing the total volume with D. I. water to 5 mL as designated by the serum producer. The pH of spiked serum is about 4.6. The procedures for SPME device preparation, extraction, back extraction, and analysis of the extract by CE have been described.5 Briefly, a stainless steel rod (o.d. 1.1 mm) was dip-coated to form membranes of dioctylphthalate- or chloroparaffin-plasticized PVC, either with or without receptor 1b after poly(vinyl chloride-covinyl acetate-co-maleic acid) was coated as the primer. Extractions were carried out by immersing the membrane-coated rod for 5 min in 3.5 mL of quiescent solutions of standard or spiked sample. Back-extraction was carried out by inserting the extraction rod into a Teflon tube containing 5 µL of phosphate (20 mM, pH 11.5) and allowing it to sit for 15 min. A portion of the back-extraction solution was vacuum injected for 4 s to a CE instrument (Isco 3850 capillary electropherograph, Isco Inc., Lincoln, NE). The separation column was a 75 µm i.d. fused silica capillary (Polymicro Technologies) with total length of 75 cm, 50 cm to the window. The separation potential was 25 kV. The separation buffer was 50 mM Tris adjusted with Tapso to pH 7.8. The volume of the extracting phase was estimated by the determination of the dioctyl phthalate in solution following redissolution of the deposited film. A rod coated with a dioctylphthalate-plasticized membrane was soaked in 3.5 mL of THF while ultrasonicating for 30 min. The concentration of dioctylphthalate in the solution was determined by CE (15 mM total borate, 50 mM SDS, pH 9.0) with UV detection. The density of the plasticized PVC membrane was estimated to be 1.0 g/mL. The concentration of the receptor in the membrane was calculated from the concentration of PVC, plasticizer, and receptor in the coating solution and the density of membrane. RESULTS AND DISCUSSION Replacing the butyryl groups in 1a with 2-ethylhexanoyl groups, 1b, is effective in increasing the solubility of the receptor. The solubility of receptor 1a in CHCl3 is about 4 mM, in dioctylphthalate it is 1.2 mM, and in chloroparaffin it is about 300 µM.18 1b (a mixture of diastereomers) is highly soluble in chloroform: solutions up to 40 wt % have been prepared with no evidence of precipitation. We have not seen evidence of its precipitation either in dioctylphthalate or chloroparaffin membranes at compositions of tens of weight percent. 1b has a very (22) Connors, K. A. Binding Constants, The Measurement of Molecular Complexes Stability; John Wiley and Sons: New York, 1987; pp 147-157.
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Table 1. Concentration Factors (pf) of Undoped Membranes and the Maximum Normalized Preconcentration Factors (npf) of Receptor-Doped Membranes in Dioctylphthalate and Chloroparaffin pf CPa butabarbital butalbital amobarbital phenobarbital pentobarbital aprobarbital secobarbital allobarbital thiopental mephobarbital a
(
sc
0.083 ( 0.004 0.046 ( 0.002 0.162 ( 0.008 0.028 ( 0.001 0.21 ( 0.01 0.049 ( 0.002 0.29 ( 0.01 0.030 ( 0.002 1.48 ( 0.05 1.17 ( 0.06
npf DOPb(
sc
0.36 ( 0. 02 0.31 ( 0.02 0.81 ( 0.03 0.159 ( 0.007 0.91 ( 0.03 0.181 ( 0.008 1.45 ( 0.07 0.093 ( 0.003 6.6 ( 0.4 2.7 ( 0.2
CP (
sc
50 ( 7 47 ( 5 42 ( 8 42 ( 4 40 ( 6 42 ( 2 31 ( 5 19 ( 4 2.5 ( 0.8 1.6 ( 0.3
DOP ( sc 25 ( 2 19 ( 1 16 ( 1 29 ( 2 15 ( 1 26 ( 1 11 ( 1 23 ( 1 1.1 ( 0.8 2.0 ( 0.2
CP, chloroparaffin. b DOP, dioctylphthalate. c s, standard deviation.
low melting point around 50 °C. Thus, it is possible, without sacrificing the ease of synthesis, to create a barbiturate receptor that is compatible with nonpolar solvents. It functions as a receptor. The formation constant for the formation of the complex with phenobarbital is 3.3 × 104 M-1 in chloroform. Therefore, we have applied it to SPME. We will define the term “preconcentration factor” (pf) as the ratio of concentration in the solution injected into the analytical instrument, in this case capillary electrophoresis, to that in the original solution. We will define the term “normalized preconcentration factor” (npf) as the ratio of the pf in a receptor-enhanced extraction to that without the receptor. As will be shown below, npf’s are receptor-concentration dependent and display a maximum. Table 1 shows values of pf and maximum npf values for the barbiturate solutes. The data show that chloroparaffin- and dioctylphthalate-plasticized PVC matrixes are poor solvents, reflecting the behavior of the pure plasticizers.18,23 However, the barbiturates can all be extracted to some degree into the receptorfree solvents. There is modest selectivity displayed by such a system based on hydrophobicity. The values of npf are all greater than unity; thus the receptordoped systems are all more efficient than solvent alone, although to various degrees. The least affected (minimum npf) barbiturates are thiopental and mephobarbital. These results are consistent with NMR binding studies on analogous macrocyclic receptors, 1c or 1d; the binding constants for thiopental and mephobarbital are relatively low16,21 K (M-1) for phenobarbital, 2.0 × 105; barbital, 6.0 × 105; mephobarbital, 6.8 × 102; and thiobarbital, 7.4 × 102. The marked difference in values of npf that reflect qualitatively the differences in the measured binding constants allows us to comment on the nature of the receptor’s activity. 1b could act as a general H-bonding cosolvent. All of the compounds in the study can act as both hydrogen bond donors and acceptors. Thiopental and mephobarbital are unique in that they do not have the native malonylurea ring. If 1b were acting as a simple cosolvent, one would not expect the values of npf to be so sensitive to molecular structure. Therefore, it is clear that 1b is acting primarily as a molecular receptor, not as a generic HBA/HBD cosolvent. (23) Valenta, J. N.; Sun, L.; Ren, Y.; Weber, S. G., Anal. Chem. 1997, 69, 34903495.
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Figure 2. Extraction of phenobarbital (X), secobarbital (]), mephobarbitral (O), and thiopental (4) with DOP membranes doped with various concentrations of receptor.
Figure 3. Extraction of phenobarbital (X), secobarbital (]), mephobarbital (O) and thiopental (4) with CP membranes doped with various concentrations of receptor.
The effectiveness of the receptor depends on the solvent.23 The chloroparaffin-based membrane displays pf values that are lower and npf values that are higher than those in dioctylphthalate. It is expected that the lower the polarity of the solvent, the more the partitioning of the polar drug will be dependent on the formation of a complex. The data in Table 1 are in agreement with that expectation. The final question concerns self-association of the receptor. Self-association is more prevalent at high concentrations than low concentrations; thus, the determination of the npf as a function of the receptor concentration should alert us to the extent of selfassociation. Figures 2 and 3 plot the npf for dioctylphthalate and chloroparaffin membranes for four barbiturates against the concentration of receptor in membrane. The data for the other six barbiturates tested are not shown on the graph but generally fall in the area between phenobarbital and secobarbital. It can be seen that, within the range of 0-0.1 M receptor, the extraction efficiency increases approximately linearly with the concentration of receptor in the extraction membrane. Indeed, it can be shown that this is expected under some reasonable assumptions. We will assume that the receptor is in excess so that the distribution of
in the system. To speak in at least semiquantitative terms to the issues just raised, a semiquantitative treatment of the NMR data is required to determine to what degree the receptor is associated at each concentration. This question is not answered easily and accurately by considering all of the possible equilibrium reactions, since the number of different “products” is vast. However, a simple but practically useful model can be derived through reasonable approximations. Each receptor has four H-bond donating functional groups (HBD, na ) 4), and six H-bond accepting functional groups (HBA, nb ) 6). Each HBA group is assumed to react oneto-one with an HBD group. Equilibrium is assumed to be achieved for the (HBA + HBD) reaction with a self-association constant K, where HA denotes all of the unbound HBD sites, B denotes all of the unbound HBA sites, B:HA denotes all of the bound sites. Figure 4. The experimental and calculated chemical shifts of H1 (square, left axis) and H2 (circle, right axis). Calculated values are shown as the solid line.
the solute does not depend on the solute concentration. We estimate the maximum solute concentration in the membrane during extraction to be a few mM, whereas the receptor concentration is on the order of 102 mM. Thus, excess receptor is a good assumption. We know that the membranes do not reach equilibrium in the extraction time allowed;5 however, the primary determinant of the extractability of these molecules of very similar molecular weight is the driving force based on the free energy difference of the solute in the feed phase and the extracting phase. Our previous data show that under the conditions used here, the extraction yield is about 1/3 that obtained at equilibrium. We make the approximation that the system is in equilibrium so that the mathematical treatment is simple. The equilibrium value of npf is a function of, Kf, the formation constant, [Rt]org, the total concentration of the receptor in the organic phase, Dc, distribution coefficient, and Φ, the phase ratio as shown in eq 1.
1 + DcΦ
npf ) (1 + Kf[Rt]org) 1 + DcΦ(1 + Kt[Rt]org)
(1)
K ) (B:HA)/(HA)(B)
There are nominally three types of basic sites and two types of acidic sites in the receptor. There are therefore six different equilibria that exist. This makes for a difficult analysis. We will assume that the acidic sites all have the same strength and so do the basic sites. It is known that the H-bonding accepting ability for pyridine and carbonyl groups in general,24 and for amide carbonyl groups in particular25-27, are similar. Thus, we are justified in assuming that the bases are the same. For the acid sites, for which we have the NMR data, we can calculate a value of the constant K for each set of data and compare the values of K to see how good our assumption is. The bound fraction of HA, Pbound, is thus expressed in terms of K, the self-association constant, na, the number of HBD per receptor, nb, the number of HBA per receptor, and Cr, the molar concentration of receptor.
Pbound ) (1/(KCr) + na + nb) ( x(1/(KCr) + na + nb)2 - 4nanb 2na (3)
Since the product of Dc (∼1)18 and the phase ratio (∼3 × 10-4) is small in our case, npf can be approximated as eq 2.
npf ≈ 1 + Kf[Rt]org
HA + B ) B:HA
Given Pbound < 1, and na ) 4, nb ) 6 for this receptor
(2)
Equation 2 shows that the slopes in Figures 2 and 3 are binding constants. Thus, the small slopes of mephobarbital and thiopental shown in Figures 2 and 3 are the direct result of small binding constants of those solutes to the receptor. The slopes for the other drugs are in the range 102-103, which is lower than one would expect,18,21 even taking into account the factor of 3 due to the lack of achieving equilibrium. Furthermore, in contradiction to the prediction of eq 2, the extraction efficiency no longer increases beyond about 0.1 M. It is likely that both the low estimate of the binding constant and the shapes of Figures 2 and 3 are explained by self-association of the receptor. To test this, 1H NMR was performed on solutions of 1b in chloroform-d. Figure 4 shows the chemical shifts of the two distinct amide protons as points. The increase in the chemical shifts with concentration indicates that self-association does exist
Pbound )
1 + 1.25 8KCr
x(
1 + 1.25 8KCr
)
2
- 1.5
(4)
The chemical shift of each of the amide protons can be expressed as a concentration weighted average of the bound and free chemical shifts as shown below.
δ ) δboundPbound + δfree(1 - Pbound)
(5)
(24) Jeffrey, G. A. An Introduction to Hydrogen Bonding; Oxford University Press: New York, 1997; p 56-78. (25) Huyskens, P. L., Luck, W. A. P., Zeegers-Huyskens, T., Eds. Intermolecular Forces An Introduction to Modern Methods and Results; Springer-Verlag: New York, 1991; p 23. (26) Kamlet, M. J.; Abboud, J_L. M.; Abraham, M. H.; Taft, R. W. J. Org. Chem. 1983, 48, 2877-2887. (27) Li, J.; Zhang, Y.; Ouyang, H.; Carr, P. W. J. Am. Chem. Soc. 1992, 114, 9813-9828.
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Table 2. Calculated Binding Constants and Chemical Shifts
K (M-1) ( sa δbound ( sa a
H1
H2
0.48 ( 0.05 8.92 ( 0.02
0.79 ( 0.06 9.92 ( 0.06
s: standard deviation.
Figure 6. Extraction of barbiturates from spiked urine. Peak assignments: (1-5) pento-, buta-, seco-, amo-, apro-barbital; (7) butalbital; (9) thiopental; (10) phenobarbital. Membranes a, c, d: plasticized with DOP and doped with receptor (90 mM); (b) plasticized with phosphate ester and without receptor. Barbiturate concentrations (a), (b) about 0.5 µM for each barbiturate except mephobarbital which is 0.2 µM, and thiopental which is 1 µM; (c) barbiturate concentrations are about 5 times those in (a) and (b); (d) blank urine.
Figure 5. The concentration of receptor with all binding sites free.
Thus eqs 4 and 5 can be combined to yield an equation for the observed chemical shift that depends on four parameters, namely, Cr, K, δbound, and δfree. The measured value of the chemical shift at the lowest receptor concentration yields δfree, and Cr is known. Then, nonlinear regression of the observed δ on Cr can be used to determine values of K and δbound. Calculated values of K and δbound from the two sets of chemical shifts are listed in Table 2. Calculated chemical shifts are shown as solid lines in Figure 4. The fits are excellent. The small difference in the self-association constants K justifies the assumption that there is not a large difference in the HBD strength between the two amide proton donors. To be concrete, the implied difference in H-bond strengths is RT ln (KH2/KH1) ), or ∼0.5RT. The foregoing calculations can be used to determine the amount of receptor that is available for binding to barbiturate. We will assume that there is a statistical mixture of receptors, some of which are H-bonded to neighbors and others that are not. We will further assume that if a receptor is not involved in HB donation, and if its pyridine groups are not involved in HB accepting (i.e., all of the six H-bond sites involved in barbiturate binding are free), then that molecule can accept a barbiturate as a guest. Recall that we have already made the assumption that the receptor is in excess. This allows us to ignore the shift in the self-association equilibrium brought about by the presence of the barbiturate. Given K as an average of the equilibrium constants from Table 2, eq 4 can be used to calculate the average probability of finding a bound state of HBD (Pbound in eq 4 is denoted as PHBD in the following to differentiate from the probability of bound state for HBA). Since each HBD must be paired with an HBA, the probability that an HBA is occupied is just 2/3PHBD because each molecule has six HBA sites for the four HBD sites that it has. The binomial distribution can then be used to calculate the fraction of the receptor population that satisfies the criteria mentioned 2150 Analytical Chemistry, Vol. 71, No. 11, June 1, 1999
Figure 7. Extraction of barbiturates from spiked bovine serum. Comparison of extraction using plasticized membranes (a) DOP doped with receptor (90 mM) and (b) phosphate ester with no receptor. (1-7) pentobarbital, butabarbital, secobarbital, amobarbital, aprobarbital, mephobarbital, butalbital; (9) thiopental. About 4.4 µM for each barbiturate except mephobarbital, which is 1.3 µM, and thiopental, which is 8.8 µM.
above. This fraction, f, is given by eq 6.
f ) (1 - PHBD)4(1 - 2/3PHBD)2
(6)
It should be noted that both binomial coefficients are unity; therefore, they do not appear explicitly in eq 6. Figure 5. shows calculated concentrations of the free receptor. First, although a quantitative comparison is not possible because the solvents are different in the extraction and NMR experiments, there is a remarkable similarity between the npf curves (Figures 2 and 3) and the curve in Figure 5. Second, self-association can be a serious problem. At the high concentration end, near 0.6 M, only about 1% of the receptor is “free”. We conclude that selfassociation exists, indeed is prevalent.
Examples of the effectiveness of the receptor-doped SPME rods are shown in Figure 6 and Figure 7 which compare the dioctylphthalate/receptor-based system to extractions based on a phosphate ester, which is a good solvent for phenobarbital.5,18,23 Electropherograms from spiked urine are shown in Figure 6, and those from spiked bovine serum are shown in Figure 7. Obviously, in these two cases the signal-to-noise ratios are improved when the receptor is added to the membrane extractor. In the urine chromatograms there are two peaks near 7.3 and 8.3 min whose concentration increases due to the receptor’s presence. We do not know what those peaks are, but their behavior shows that there may be compounds in complex samples that cannot be discriminated against with a receptor-doped solvent. CONCLUSION The concept of using a doped, poor, nonvolatile solvent for the extraction of organics from aqueous samples is feasible. There are many advantages to this. There are no volatile organic solvents in the extraction, and there is some molecular selectivity in the
extraction. The advantage of molecular selectivity comes from selectively preconcentrating analytes. We cannot say with confidence that the molecular selectivity is absolute, however. The extraction devices still contain a solvent, and although it is poor, it can still dissolve solutes that do not bind to the receptor. There may be other compounds that interact with the barbiturate receptor. Nonetheless, if the complexity of a sample can be reduced before its entry into a separations/detection system, then lower detection limits and/or higher speed analysis will result. ACKNOWLEDGMENT We thank the NSF for financial support through Grant CHE9710213.
Received for review May 27, 1998. Accepted March 10, 1999. AC980587O
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