Anal. Chem. 1994,66, 2397-2403
Enhanced Extraction of Phenobarbital from Serum with a Designed Artificial Receptor Jane N. Valenta, Robert P. Dixon,t Andrew D. Hamilton, and Stephen G. Weber' Department of Chemistry, University of Pittsburgh, Pittsburgh, Pennsylvania 15260
The primary goal of this work was to determine whether artificial receptors that function on the basis of molecular recognition have analytical capabilities. As an example of such a receptor, we have chosen one directed toward barbiturates. Chloroform enriched with this artificial receptor (1 mM) can extract more than 90% of the phenobarbital from a 20 rM phenobarbital solutionin human control serum using a volume ratio (organic/serum) as small as 0.5. In the absence of this receptor, the volume ratio must be greater than 10 to achieve similar extraction efficiencies. In addition to volume ratio, the role of pH, receptor concentration, and solvent type are discussed. The experimental results are found to be in good agreement with predictions based on chemical equilibria. Through the use of this and other similar receptors, the amount of organic solvent used in extractions can be minimized. Liquid-liquid 'Jand solid-phase3extractionshave been used extensively in chromatography for sample preparation. Solvent extraction is a convenient method for isolating an analyte or group of analytes from a complex matrix such as blood serum. The extracting solvent offers a very limited degree of selectivityand so it is not uncommon to find interfering species in the extract. The lower the degree of selectivity of the extraction step, the greater the demands placed on the selectivity of the ensuing chromatographic determination. From a practical standpoint, an extraction should extract selectively the analyte of interest and concentrate it in a minimum number of steps. Historically, the extraction of ions has benefited from the use of transition metal chelating agents4 that can selectively extract metal cations from aqueous solution. Recently Bartsch and co-workers- have turned attention to the more difficult problem of alkali and alkaline earth metal ion extractions. A series of crown ether derivatives was synthesized and systematically studied for their ability to extract alkali and alkaline earth metal cations from aqueous solution into chl~roform.~-~ There has been very little effort directed toward molecularly selective extractions. Antibodies have been used in the Current address: Department of Chemistry, Northwestern University, Evanston, IL 60208. (1) Poolc, S. K.; D a n , T. A,; Oudsema, J. W.; Poole, C. F. Anal. Chim. Acta 1990, 236,3. (2) Furton, K. 0.; Rein, J. AMI. Chim. Actu 1990, 236, 99. (3) Krishnan, T. R.; Ibraham, I. J. Pharm. Biomed. Anal. 1994, 1.7, 287. (4) Fritz, J. S.;Schenck, G. H. In Quanrirariue Analyrical Chemistry; Allyn and Bacon, Inc.: Boston, MA 1979; pp 425433. (5) Strzelbicki, J.; Bartsch, R. A. Anal. Chem. 1981, 53, 1894. (6) Charewicz, W. A.; Hw,G. S.;Bartsch, R. A. Anal. Chem. 1982,54,2094. (7) Walkowiak, W.; Kang, S.I.; Stewart, L. E.;Ndip, G.; Bartsch. R. A. Anal. Chem. 1990,62,2022. (8) Walkowiak, W.; Ndip, 0.; Dcsai, D. H.; Lee, H. K.; Bartsch, R. A. Anal. Chem. 1992.64, 1685. f
000~2700/94/036&2397$04.5~l0 Q 1994 Amerlcan Chemical Society
extraction of drugs9 for example. Artificial receptors offer a flexible strategy for small-molecule extractions. Though the selective extraction of butylamines,I0 creatinine," and the amino acids phenylalanine, tryptophan, and tyrosine12 have been demonstrated, there have been to our knowledge no systematic investigations of the molecularly selective extraction of any organic species. The system that we are currently investigating represents the larger class of artificial receptors that are based on hydrogen bond-directed molecular r e c o g n i t i ~ n . ~In ~ -this ~~ work, an enhanced extraction medium has been developed basedon an artificial receptor molecule1*that binds specifically to 5,5-disubstituted barbiturates (Figure 1). The cavity of the receptor molecule is lined with hydrogen-bonding groups that are complementary to the hydrogen-bonding groups of barbiturates. The binding interactionsof the receptor-barbital complex have been evaluated in CDCl3 by lH NMR and a value of 2.08 X IO4 M-I has been formation constant (Kf) determined.18 This formation constant is large enough that practical application of the receptor may be considered. The purpose of this paper is to investigate the analytical capabilitiesof a synthetic receptor for the enhanced extraction of phenobarbital from serum. In particular, the use of such receptors should make extractions possible with less organic solvents. There are several questions that arise in conjunction with this, or any other attempt to use hydrogen bond-directed molecular recognition in this manner. This barbiturate receptor is relatively polar. Binding to barbiturates is favored in nonpolar solvents and weakened in polar solvents. Thus, it is important to determine whether a practically useful concentration of a rather polar receptor can be achieved in an extraction medium that favors binding. In addition to solubility being an issue, the presence of additional hydrogenbonding substrates (e.g., water and serum components) which may interfere with phenobarbital-receptor binding is a concern as well. Another aspect of the extraction that needs to be addressed is pH. Traditionally, the adjustment of pH of the aqueous sample has been used to control selectivity but the (9) Miyairi,S.;Shimada,H.;Awata,N.;Goto, J.;Nambara,T. T. Phann.Eiomed. Anal. 1994, 12, 389. (10) Chang. S . K.; Jang, M.J.; Han, S. Y.;Lee, J. H.;Kang, M. H.; No, K. T. Chem. Leli. 1992,10, 1937. (1 1) Buhlmann, P.; Badertscher, M.; Simon, W. Terruhedron 1993,49, 595. (12) Rebck, J., Jr.; Nemeth, D. J. Am. Chem. Soc. 1985, 107, 6738. (13) Lchn, J. M.Science 1985, 227. 849. (14) Cram, D. J. Agnew. Chem., Inr. Ed. Engl. 1988. 27, 1009. (15) IzatLR. M.;Bradshaw, J. S.;Pawlak,K.; Bruening,R. L.;Tarbet,B. J. Chem. Rev. 1992, 92, 1261. (16) Rebek, J., Jr.; Askew, B.; Killoran, M.;Nemeth, D.;Lin, F.T. J. Am. Chem. Sm. 1987,109. 2426. (17) Hamilton, A. D. In Advances in Supramolecular Chemistry; Gokel, G. W., Ed.; JAI Press, Inc.: Greenwich, CT, 1990; Vol. 1. pp 1-64. (18) Chang, S.K.; Hamilton, A. D. J. Am. Chem. Soc. 1988. 110, 1318.
AnaWaiChemisby, Voi. 68, No. 14, Ju& 15, 1994 2587
4
0.4
0.2 0
c 0 0
e 51 n
o
U
-0.2 Flgure 1. Structure of the barMhrate receptor and the hydrogenbondinglnteractkns between barb#urate receptorand phembrbital.
effects of pH on binding are not known. This paper not only addresses these issues but it evaluates the experimental results in light of equilibrium considerations.
EXPERIMENTAL SECTION Binding Studies. Reagents. Spectrophotometric grades of chloroform and methylene chloride were purchased from Mallinckrodt SpecialtyChemicals Co. (Paris, KY)and Aldrich Chemical Co. (Milwaukee, WI), respectively. Barbital, amobarbital, and phenobarbital were purchased from Sigma Chemical Co. (St. Louis, MO). The details of the barbiturate receptor synthesis have been described elsewhere.’* The barbiturate receptor solutions were placed in an ultrasonicator for 10 min to hasten the dissolution of the receptor in chloroform. Apparatus. UV absorbance measurements were made using either an IBM 9420 UV-vis spectrophotometer or Hewlett-Packard 8450A diode array spectrophotometer. Quartz cuvettes with path lengths of 0.1 and 1.0 cm were purchased from Fisher Scientific (Pittsburgh, PA). Procedure. Two different experimental approaches were for receptor pursued to determine the dissociation constant (4) complexesinvolving barbital, amobarbital,and phenobarbital. In the first set of experiments, the concentration of receptor was held constant while the concentrationof barbiturate was varied. For these experiments, 1.0-cm path length quartz cells were used and the total receptor concentration was held constant between 40 and 75 pM. In the second set of experiments, a series of dilutions was made in which the concentrations of receptor and barbiturate were very similar. Concentrationsranged from 0 to 505 pM for the barbiturates and the receptor. Linear calibration plots for receptor and receptor-phenobarbital solutions were obtained for the concentration ranges studied. At concentrationsbeyond 121pM, spectrophotometricquartz cells of 0.1-cm path length were necessary. In the wavelength range where there is no interference from the barbiturateabsorption (A- z 240nm), there is a large absorption band at 302 nm in the receptor. This absorption band shifts to 305 nm upon formation of the receptor-substrate complex. The maximum difference in absorptivities of the receptor and the receptor-substrate complex is 318 nm. The concentration of the RS complex was determined by directly measuring absorbancechanges at either 3 16 or 3 18 nm or by measuring the absorbancechanges occurring at 318 and 290 nm by difference spectroscopy. Representative UV spectra for difference measurements are 2998
AnaWcal Ch8mktry, Vol. 66, No. 14, JldL 15, 1994
250
300 350 Wavelength (nm)
-
Flgwe 2. Rep”tathmWq”forasoMonoocitrdnhg 6.90319 pM 1and 65 N reoeqtor In mahyknr cMor#.. T-1.
-hYolw-lor
substrate
solvent
barbital barbital amobarbital amobarbital phenobarbital phenobarbital
chloroform methyknc chloride chloroform methylene chloride chloroform methylene chloride
AellS
Agio
hcyllb
(L/molcm) (L/molcm) (L/molcm) 4780 6260 4820 6030 5230
5910
5410 6130
8310 lo400 8640 lo400 8880 10700
shown in Figure 2. The molar absorptivities for the receptor at 3 18 nm in chloroform and methylene chloride are 12 700 and 12 300 L mol-’ cm-1, respectively. Thus for the singlewavelength measurement, the absorbance must be corrected for the concentration of free receptor in solution. The concentration of the RS complex can be calculated for the single wavelength measurements by using [RS1 = (AT1 - Afl)/(bAEAl)
(1)
where [RS] is the concentration of receptor-barbiturate complex, A:, is the baseline-corrected absorbance of a solution containingthe barbiturate receptor, barbiturate, and receptor-barbiturate complex at XI, b is the path length, A fi is the baseline-corrected absorbance of a solution containingonlythebarbituratereceptoratXl,and Aeu isthedifference in molar absorptivities for the barbiturate-receptor complex and barbiturate receptor (cw - E R ) at XI. The molar absorptivity ( E M ) for each of the barbiturate-receptor complexes is determined from spectroscopic studies in which the barbiturate substrate is in sufficient exccss compared to the receptor concentration such that all of the receptor present in solution is converted to receptor-barbiturate complex. ~ be found in Table 1. For the dualValues of A E Acan wavelength measurements [RS]is calculated as,
where AAmxminis the difference between the absorbance at 318 nm and that at 290 nm (A318 - A ~ w )and , A t u n is the
difference in molar absorptivities of the RS complex at 3 18 and 290 nm (~318-~290).Table 1 lists Acmaxminvaluesobtained from binding studies carried out in chloroform and methylene chloride. SpectroscopicStudies. Reagents. Control serum Type I-A was purchased from Sigma Chemical Co. The serum was reconstituted as directed. Spectral grade chloroform was purchased from Mallinckrodt Specialty Chemicals Co. The buffer solutions for the pH range 3-10 were prepared daily with water passed through a Milli-Q water purification system (Millipore, Bedford, MA). The buffers for pH 3-8 (citrate phosphate), pH 9 (borax-HCl), and pH 10 (borax-NaOH) were prepared according to ref 19. Citric acid (Aldrich Chemical Co.), sodium phosphate, dibasic (EM Science, Gibbstown, NJ), sodium borate, 10-Hydrate (J. T. Baker Inc., Phillipsburg, NJ), sodium hydroxide, and hydrochloric acid (MallinckrodtSpecialty Chemicals Co.) were used as received. Apparatus. The UV absorbancemeasurements were made using either an IBM 9420 UV-VIS spectrophotometer or Hewlett-Packard 8450A diode array spectrophotometer. Spectrophotometric quartz cuvettes with a path length of 1.O cm were purchased from Fisher Scientific. Procedure. Aqueous solutionsof phenobarbital (zl00pM) were transferred to a scintillation vial containing an equal volume of extracting solvent (chloroform,200 pM barbiturate receptor-chloroform, or 1 mM barbiturate receptor-chloroform). The contents of the vials were mixed gently for a few seconds using a Vortex-Genie (speed control 1, Scientific Industries, Bohemia, NY), vented, and placed on a shaker (setting 4, Eberbach Corp., Ann Arbor, MI) for 30 min. The samples were removed from the shaker, and after a short period of time, the layers separated. The aqueous and organic layers were then transferred to clean vials. All of the extractions were carried out at ambient temperature (22.7 f 1.76 "C) and examined in duplicate. Measurements. The UV absorbance spectra of barbiturates are pH dependent. Aqueous solutions of phenobarbital were examined by UV spectroscopy prior to and after each extraction as a function of pH. The percentage of phenobarbital extracted at each pH value was based on changes in absorbance at a predetermined analytical wavelength. All UV measurements were baseline corrected. Chromatographic Studies. Reagents. The mobile-phase buffer was prepared from stock solutionsof 0.50 M potassium phosphate, monobasic (EM Science) and 0.45 M phosphoric acid (EM Science). The pH of the buffer solution wasadjusted to pH 4.3 using a 1 M NaOH solution. The mobile phase consisted of (v/v 9%) 65% 20 mM phosphate buffer-21% methanol (Mallinckrodt Chemical Specialty Co.)-14% acetonitrile (EM Science). All of the solvents were filtered prior to use. The organic solvents were filtered through Teflon filters (pore size 0.45 pm), while the aqueous solvents were filtered through cellulose ester filters (0.45-pm pore size). The aqueous solutions were prepared daily using Milli-Q water. Apparatus. The chromatographic system consisted of a Waters 600E pump, Hewlett-Packard 1050 autosampler, an Alltech precolumn (Brownlee RP-18 New Guard Cartridge), a Hypersil ODS CIS 15 X 4.6 (cm X mm) column, 5-pm (19) Lunge's Handbook of Chemistry; Dean,J. A., Ed.; McGraw-Hill New York, 1985; pp 69-82.
Book Co.:
packing, and a Waters 990 diode array detector. Data were acquired using a Waters 990+Version 6.22A data acquisition program. Experiments were carried out at ambient temperature. The chromatographic conditions were as follows: wavelength of detection for phenobarbital 196 nm, flow rate 1.0 mL min-1, and injection volume 50 pL. The average chromatographic run time was 10 min. Procedure. Twenty micromolar solutions of phenobarbital in aqueous buffer (pH 8 , 2 X M KH2PO4-5 X 10-4 M NaOH) and serum were extracted with chloroform and 1 mM receptor-chloroform solutions, respectively. The volume ratio (VorJVaq) ranged from 0.25 to 10. The extraction procedure was carried out in an identical manner as described above in the spectroscopic studies procedure section with the exception that the extraction time was increased from 30 min to 1 h. The serum sampleswere centrifuged (Adams analytical centrifuge, Clay-Adams, Inc., New York, NY) after each extraction in order to completely separate the two layers. The aqueous and organic Iayers were placed in separate test tubes. The aqueous layers for pH 8 solutions were filtered and examined directly by reversed-phase HPLC. The organic extracts (of known volume) for both the pH 8 solutions and serum sampleswere placed on a reaction block set (temperature setting 55 OC, nitrogen purge) for 5 min; the dried residue was then reconstituted with mobile phase, placed in an ultrasonicator for a few minutes to resolubilize completely the residue, filtered, and examined by reversed-phase HPLC. The chromatographic procedure for phenobarbital determination was adapted and modified from a method previously described in the literature by Gerson et a1.20 Measurement. The percentage of phenobarbital extracted was calculated on the basis of the changes in peak height prior to and after each extraction. Phenobarbital standards were prepared daily in mobile phase and placed randomly among the samples on the autosampler. A calibration curve was prepared for each set of runs. There were day-to-day variations in retention times, but these variations on the average were less than 30 s.
RESULTS AND DISCUSSION Equilibrium Considerations. The dissociation constants (Kd)for the receptor-barbiturate complexes in chloroform and methylene chloride were determined using the following equation:z1
(R, + S,)= (R,S,)/[RSI+ [RSI - Kd
(3)
where Rt is the total receptor concentration, St is the total barbiturate concentration, and [RS] is the concentration of barbiturate complex present in solution. Linear regression of [RS] in eq 3 leads to Kd. A (R, S,) on (R&)/[RS] representative example of this approach is given in Figure 3. Table 2 lists Kd values for barbital, amobarbital, and phenobarbital in chloroform and methylene chloride. These data show that binding is more favorablein methylenechloride than chloroform. One plausible explanation is that chloroform is more acidic than methylene chloride. As such, it is more
+
+
(20) Gemon, B.;Flell, F.;Chan, S.Clin. Chem. 1984, 30, 105. (21) Connors. K.A. InBindingCmranrs, TheMcaturcme~ofMolcculorComplex Srabiliry; John Wiley and Sons: New York. 1987; pp 147-157.
AmWCal Chemistof, Vol. 66,No. 14, July 15, lQQ4
2389
001
r
-
0
0 0
0008
0
-
0 0 0
Y 3
L
0
'i; 0006 -
0 0
b 4 .
cn
+
z
0 0
0004 -
0
I
wls
0 0
0002
-
0
01
I
#
I
Flour@4. Schematic &gram of an e x t " of p h m h h h l In the prbssnce of barbiturate receptor. substrate barbital amobarbital phenobarbital
chloroform
methylene chloride
4.67 i 0.670 X itsb 2.49 i 0.503 X lo-% 3.70 i 0.328 X lo-% 2.18 t 0.153 X lo-" 1.99 t 0.400X
4.80 f 0.236 X l e 1.14 t 0.816 X 10-k
defined by eq 5 , where CP is the volume phase ratio (Vowdc/
1.20 t 0.440 X 10-sb
q = I/@,@
(5)
0 fsdenotarstandard deviations. b Denoteranstant [R] experimental approach. c Denotes dilution wperimcntal approach.
competetive in its interactions with substrate and receptor than methylene chloride. A more thorough investigation of solvent effects on binding is warranted, and this investigation has been initiated and will be reported in the future. In a simple extraction, phenobarbital equilibrates between an aqueous and an organic phase. The completeness of the extraction depends on two equilibria, the acid dissociation of phenobarbital and partitioning of the neutral phenobarbital between the two phases. The partition coefficient, Kp refers to the partitioning of the neutral formof phenobarbital (SH2) between the two phases and does not account for the ionized forms of phenobarbital (SH-and S2-). More relevant to the extraction is the the distribution of all forms of phenobarbital between the two phases. The distribution coefficient, Dc, is defined in eq4, where KaI is the first acid dissociationconstant 0,= Kp/(l + K,,/[H+I)
+ 1) = (1 + K,,/[H+])/(K,@+ 1 + K,/[H+I)
(4)
of phenobarbital. Note that eq 4 does not take into account the second acid dissociation constant of phenobarbital. The pKa values for phenobarbital22are 7.3 and 11.8, respectively. The second pK, value has been omitted from all theoreticel treatments because all experiments were carried out at pH values less than 10, and therefore, the conrxntrotion of S2-is always negligible. The completenessof the exuaction is based on the fraction of phenobarbital remaining ( q ) in the aqueous phase. It is (22) Chao, M.K.C.; Albert, K.S.;F U ,S.A. In Ana&tlca/ PtoJlka of Drug Svbshinces; plorey, K.,Ed.;Academic h, IN.: N e w York, 1978; Vol. 7, p 374.
Vqw). Thus, the amount of phenobarbital extracted from the aqueous layer for a simple extraction depends on pH explicitly, solvent type through Kp,and phase volumes explicidy The system becomes slightly more complex with the introductionof an artificial receptor moleculefor phenobarbital into the overall extraction scheme (Flgure 4). The enhanced extraction efficiency arises from the binding interactions between phenobarbital and the barbiturate receptor. If the total number of moles of receptor (R) is in excess, there is no partitioning of the phenobarbital-barbiturate receptor complex between the two phases, and the receptor is insoluble in the aqueous phase, then the simple relationship, eq 6, can be
.
4 = 1/[W (1 + WtlorJ + 11
(6)
derived in which Kr is the formation constant for the phenobarbital-barbiturate receptor complex (Kf= &-I) and [Rt] is the total concentrationof barbiturate receptor present in the organic phase. Equation 6 not only takes into account the dependence of the extraction on pH, solvent type, and phase volumes but also accounts for the influence of receptor concentration and the binding constant for the complex. In the followingsections, this equation will be used to predict the efficiency of the extraction for a given set of experimental con&thns. Distribution coefficients (De) values were calculated for pHs ranging from 2 to l l by taking the Dc value of 4.16 obtained from the literature22at pH 3.4 and using eq 4. A formation constant (Kf) value of 4.59 X 104 M-1 was determined from the phenobarbital-barbiturate receptor binding studies carried out in our laboratory (Table 2). Equations 5 and 6 were used to calculate the theoretical fraction of phenobarbital remaining ( q ) after each extraction
c
I
z
I
100
m
Y
X
W
75
CI 3
P L m
n
E
2 00
250
300 Wavelenq t h (nm)
50
PH
350
Flgure 5. Qualitative demonstration by UV spectroscopy of p h a nobarbital extraction: aqueous layer prior to extraction (a); aqueous layer after extractbn with chlorofonn(b); aqueous layer after extractbn wlth 890 pM barbltvate receptor (c). Conditkns: ink1 [phenobarbital], = 105 pM; CP = 1.0.
Flguo8. Comparisonof experlmentaland theoretical data as a functbn of receptor concentration. Mot of percentage of phenobarbitalvs p H 200 pM receptor @); 1 mM receptor (A);theory (solM chloroform (0); line). Conditions: lnltlal [phenobarbitai]., = =lo0 pM; CP = 1; K, = 4.59 x 104 MI. I
"
'
"
O . O 5 t With Receptor
as a function of pH, receptor concentration, volume phase ratio, and solvent type. It is important to note that there are no adjustable parameters in eq 6. Values are either obtained from the literature or from experiments carried out in our laboratory. Qualitative View. A qualitative demonstration of phenobarbital extraction is given in Figure 5 . Figure 5 shows a fairly complete extraction of phenobarbital from aqueous solution at pH 8 in the presence of receptor (curve c) while only about half of the phenobarbital could be extracted with chloroform (curve b). Curve a represents the concentration of phenobarbital present initially in the aqueous layer prior to extraction. The presence of receptor in the extracting solvent, chloroform, has a remarkable effect on the quantity of phenobarbital extracted from aqueous solution. Curve c also shows that only a small fraction, about 0.1%, of the receptor is extracted into the aqueous phase (peak near 300 nm). Quantitative Studies: Influence of pH and Receptor Concentration. The efficiency of the extraction was determined based on absorbance changes prior to and after each extraction as a function of pH and receptor concentration. The theoretical and experimental percentages of phenobarbital extracted as a function of pH are given in Figure 6. The solid lines represent the predicted results in the absence and presence of 0.2and 1 mM receptor (eq 6) while the experimental results are represented by symbols. The results for the chloroform extractions in the absence of barbiturate receptor are interpreted fairly straightforwardly because the first acid P M (pKa = dissociation constant for phenobarbital is 5 X 1 7.3). As the pH of the solution increases, the solubility of phenobarbital in the aqueousphase increases through increased ionization. This limits the amount of phenobarbital available in its neutral form. However, when the organic phase contains 1 mM barbiturate receptor, complex formation stabilizes the neutral form of the solute. This results in the appearance that the pKa shifts to a more basic value. From a quantitative standpoint, at pH values of 17,the ratio of phenobarbital extracted in the presence and absence of 1 mM barbiturate
0
5
Without Receptor
10
0
5
10
Time ( m i n ) Flgure 7. Qualitative demonstratlon by RP-HPLC of phenoberbital extractionfromserum.Thedlrect Injectionofchlorofonnleyersfdkwlng extractbn. Condftlons: ink1 [phenobarbital]-,, = 129pM; [receptor] = 1 mM; CP = 1; Injectionvolume 10 pL; flow rate 1 mL mln-l; detection wavelength 196 nm.
receptor is 1.2:l. At these pH values, the extraction is only slightly enhanced in the presence of receptor because the solvent alone extracts a significant amount of the barbiturate. On the other hand, at pH values greater than 7, the effect of the receptor is more pronounced. As expected, a higher concentration of receptor leads to higher extraction yields. There is an advantage to having a wider range of pH over which theextraction is acceptablebecausethechanceof finding selectivity against interfering organic species is more likely the wider the available pH window. This is an important practical consideration when one is analyzing for trace levels of phenobarbital in serum. The theoretical and experimental results are in very good agreement with one another in Figure 6. QuantitativeStudies: Influence of Phase Ratio. Reversedphase high-performance liquid chromatography was used to investigate the effects of volume on extraction efficiency. Representative chromatograms for the organic extracts of serum in the presence and absence of receptor are given in Figure 7. In these chromatograms, two distinct peaks are present; one peak represents phenobarbital while the other represents one or more serum interferences. The chromatograms demonstrate that the magnitude of the phenobarbital peak is influenced by the presence of receptor, while the magnitude of the interfering peak is not. This illustrates the selectivity of the receptor for phenobarbital over this (these) particular interference(s) arising from serum. Ana&ticaiChemistty, Vol. 60, No. 14, Ju& 15, 1994
2401
wtnout Receptor
With Receptor
0.1
t
i-5L E
n o0
2
A
volume Ratlo
6
8
- Vorg/VW
10
O 0
2
1
6
Volume Ratio
8
1
0L
- Vorg/Vaq
A larger volume of extracting solvent than sample is necessary to remove (on the order of 10:l)23J4an appreciable quantityof phenobarbital fromsemm. In this work, wewanted to demonstrate that this was not the case when receptorenriched solvent was used. Twenty micromolar solutions of phenobarbital (pH 8 buffer or serum) were prepared and extractions were camed out with chloroform and 1 mM rcceptor-chloroform using volume ratios ranging from 0.25 to 10. A buffer of pH 8 was chosen for two reasons: (1) experiments had shown that at pH values below 7 the receptor had minimal effect on extraction efficiency, and (2) the pH of the serum samples upon reconstitution was 8. Figure 8 represents the extraction results for aqueous and serum solutions of phenobarbital in the absence and presence of receptor. The solid lines represent the predicted percentages of phenobarbital extracted as a function of volume ratio. The symbols represent the experimntal results. In the case of the chloroform extractions (Figure 8a), a volume ratio greater than or equal to to 10 is nectssary to extract an appreciable quantity ( ~ 8 0 % )of phenobarbital from aqueous or serum solutions. On the other hand, for extractions carried out in the presence of 1 mM receptor (Figure 8b), a volume ratio of 0.5 was more than sufficient to extract at least 90%of the phenobarbitalpresent in the original sample. Also, Figure 8a shows that it is more difficult to extract phenobarbital in the absence of receptor from serum, possibly due to interactions between phenobarbital and Serum proteins. In Figure 8b,note the shape of the experimental results for pH 8 buffer extractions involving 1 mM barbiturate receptor. The data points for this curve represent RP-HPLC results for both the aqueous and organic layers following extraction. A considerable discrepancy between the theoretical and experimental results is evident for a few points at volume ratios less than 1. In particular, four data points stand out. Three of these four data points represent aqueous extracts obtained on (23) Rpdoyuuh, 1. N. HPLC in Clinical Chewdstv, Academic Rcss: New Yalr. 1976: p~ 242-260. (24) U t , R.C. AncrlytluJ pmvdwr for TkrapmUfcDrug Monitoring and E m c " y Toxlcologv, 2nd ed.: P!3G Publbhing Co.,Inc.: Littleton, MA, 1987; pp 26-39.
the same day while the other data point represents the organic extract obtained on a different day. At this point in time, we have rationalized this discrepancy in terms of incomplete extractions. At volume ratios less than 1, the total surface area of the organic layer is much less than the total surface area of the aqueous layer. Thus, the extraction may require much longer extraction times than 1 h or perhapp require a more vigorous shaking to ensure complete mixing of the two layers. A similar discrepancy can be seen in Figure 8a at volume ratios less than 1. SohenQ. Solvent was another important experimental consideration. It was felt that use of a hydrophobic solvent such a8 toluene in place of chloroform would limit theaumber of interferences arising from serum in the extract. Experimentally, this was found to be true and is shown spectrompically in Figure 9. In Figure 9, serum samples extracted with chloroform were found to contain a higher absorbance from interfering species than those extracted with toluene. While certainly the level of the interference from nonanalyte molecules in a sample depends on the analytical technique used, it is clear that there is less colored material extracted by toluene than by chloroform. In addition to minimizing polar interference, we alsoanticipated that smaller quantities of phenobarbital wouM be extracted (in the presence and absence of receptor) for a number of reasons. First, phenobarbital has a very limited solubility in toluene. The literatun reports distribution coeffhent values for phenobarbital of 0.552 (toluene) vs 2.35 (chloroform) at pH 7.4.u Second, the solubility of the barbiturate receptor is extremely limited in toluene (z5 vs r 5 0 0 mg/L in chloroform). The receptor's limited solubility in toluene has prevented us from taking a closer experimentalexamination of receptorenriched toluene. Thus, the receptor molecule will require synthetic modification if it is to be employed successfully in toluene. However, we can gain some insight from eq 6. In theory, as the solubility of the modified receptor increases in toluene, one can expect extraction efficiencies equal to those of receptor-enriched chloroform. However, as Sten in theoretical plot given in Figure 10,extractions camed out between pH 8 and 10 with receptor-enriched toluene will require a much higher concentration of receptor to achieve extraction efficiencies equal to those of receptor-enriched chloroform. For the sake of illustration, we have taken the formation
w
75
5 4 o
25
i\
PH Figure 10. Comparison of theoretical results for receptor-enrlched solvent from equation 6 to Illustrate the effects of changing solvent. A plot of percentage of phenobarbital extracted vs p H receptorenriched chloroform (0);receptor-enriched toluene (A).Conditions: = 4.59 X lo4 M-l; [receptor] = 1 mM; @ = 1.
artificial receptor (1 mM) can extract more than 90% of the phenobarbital from a 20 pM phenobarbital solution in human control serum using a volume ratio (organic/serum) as small as 0.5. In the absence of this receptor, the volume ratio must be greater than 10 to achieve similar extraction efficiencies. This work clearly demonstrates the selectivity of the barbiturate receptor for phenobarbital over serum interferences. Our initial concerns regarding pH and water were found to be unwarranted; however, receptor solubility still remains a key issue. The future of this artificial receptor depends principally on synthetic modification to improve receptor solubility. The building of even better artificial receptors with even greater solubilities in nonpolar solvents such as toluene will produce even moredramatic extraction results as a function of [receptor], pH, volume ratio, and solvent type.
ACKNOWLEDGMENT constant for the phenobarbital-barbiturate receptor complex in toluene to be equal to its value in chloroform. Theoretical calculations indicate that the concentration of the receptor in toluene will have to be on the order of 8 mM in toluene, compared to 1 mM in chloroform, to achieve similar extraction efficiencies between pH 8 and 10. This increase in receptor concentration is necessary to overcome the differences in the distribution coefficient, D,.
CONCLUSIONS Receptor-enriched solvent extracts phenobarbital from serum with high efficiency. Chloroform enriched with this
This work was supported by the Material Research Center at the University of Pittsburgh, which is funded through AFOSR and the Office of Naval Research (to A.D.H.). We express our gratitude to PPG Industries, Inc., for their educational support (to J.N.V.) and Gerald Feulmer (PPG Industries, Inc.) for his assistancewith the HPLC experiments.
Received for review January 3, 1994. Accepted April 20, 1994."
Abstract published in Aduance ACS Abstracts, June 1, 1994.
AnalyticalChemlstry, Vol. 66, No. 14, Ju& 15, 1994
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