2822
Anal. Chem. 1984, 56,2822-2825
not Fe(CN),", and the former does not react with molybdate ion so that no change of color is expected. In this case, it is suggested that prussian blue is formed by the reaction of Fe3+ with Fe(CN)63-as shown in eq 5-1-5-4. In Figure 5, an isosbestic point was observed near 520 nm, indicating the presence of an equilibrium reaction in the system. It is interesting that such a reaction is proceeding at the surface region of the spot under light illumination. This fact implies that the photoacoustic spectroscopy is a useful tool to trace and investigate photochemical and surface reaction.
ACKNOWLEDGMENT We thank S. Ikeda and I. Watanabe of Osaka University and Y. Yokoyama of Technological University of Nagaoka for the useful suggestions regarding the assembly of the photoacoustic spectrometer. Registry No. K,Fe(CN),, 13746-66-2;K4Fe(CN)G, 13943-58-3; FeCl,, 7705-08-0; FeC12, 7758-94-3; Prussian Blue, 12240-15-2; Turnbull's Blue, 65505-26-2; ferric ferricyanide, 14433-93-3.
LITERATURE CITED (1) Treadwell, F. P. "Analytical Chemlstry"; (translated by Hall, W. T.) Wiley: New York, 1935; Vol. 1.
Takagi, S."Quantitative Analysis"; Nankodo: Tokyo, 1981: Vol. 11; p 345. Feigl, F. "Chemistry of Specific, Selective and Sensitive Reactions"; Academic Press: New York, 1949. Levi G. Chim. Ind. Appl. 1925, 7, 410. Weiser, H. B.; Miliigan, W. 0.; Bates, J. B. J . Phys. Cbm. 1942, 46, 99. Ludi, A.; Gudel, H. U. Struct. Bonding (Berlin) 1973, 14, 1. Felgl, F. "Spot Tests in Inorganic Analysis"; Elsevier: Amsterdam, 1958. Feigl, F; Suter, H. A. Chemlst-Analyst 1943, 32,4. Mortlmer, R. J.; Rosseinsky, D. R. J. Electroanal. Chem. 1983, 151, 133. Ellis, D.; Eckhoff, M.; Neff, V. D. J. Phys. Chem. 1081, 85, 1225. Itaya, K.; Shibayama, K.; Akahoshi, H.; Toshima, S. J . Appl. Phys. 1982, 53, 804. Itaya, K.; Akahoshi, H.; Toshima, S.J. Nectrochem. SOC. 1982, 129, 1498. Itaya, K.; Ataka, T.; Toshima, S. J . Am. Chem. SOC. 1982, 104, 4767. Kawamoto, S.;Yokoyama, Y.; Ikeda, S.Bull. Chem. SOC.Jpn. 1980, 53, 391. Ikeda, S.; Murakami, Y.; Akatsuka, K. Chem. Lett. 1981, 363. Rosencwalg, A.; Hall, S.S.Anal. Chem. 1975, 47,548. Robin, M. B. Inorg. Chem. 1062, 1 , 337. Robin, M. B.; Day, P. Adv. Inorg . Chem. Radiochem. 1987, 10, 247. Wenger, P.;Duckert, R. Helv. Chim. Acta 1044, 27, 757. Voglar, A.; Adamson, A. W. J. Phys. Chem. 1070, 74,67.
RECEIVED for review April 25,1984. Accepted July 30, 1984.
Solute- Induced Circular Dichroism: Complexation of Achiral Drugs with Cyclodextrin Soon M. Han and Neil Purdie* Chemistry Department, Oklahoma State University, Stillwater, Oklahoma 74078
The formation constants for the association of eight achiral drug molecules with P-cyclodextrln in aqueous media have been determined by using data from the induced circular dichroism spectra for the drugs. The data have also been used in the determlnatlon of meperidine (Demerol) In a dlspensary product.
The ability of @-cyclodextrinto act as a host in complexing a wide variety of other molecules in aqueous solutions has been recognized for many years and recently reviewed ( I , 2). The nature of the interaction is fairly well understood, and the process has been successfully applied to enhance the aqueous solubility of organic molecules (I,2), as an enzymatic model system (3),and to effect the chromatographic separation of structural isomers (2, 4 ) . The interactions have been the subject of a number of thermodynamic studies ( I , 2 , 5 , 6 ) and at least one indepth theoretical study (7). From this wealth of information i t is understood that the center of the cyclic oligosaccharide is hydrophobic, making it accessible to nonpolar, usually aromatic, molecular moieties. It is generally believed also that the structure of the guest molecule external to the interaction site has little to no influence on the process or the physical properties of the complex (1-3). As a potential analytical reagent @-cyclodextrin and its aqueous solutions are stable, although the solubility is somewhat limited. It is available in relatively high purity and
is inexpensive. The property that we wished to exploit in this work is the ability of the molecule to induce chirality into an achiral guest molecule. If the guest contains a chromophore, the interaction produces a complex which will give a circular dichroism (CD) spectrum. The spectra could be used for drug identification and as a method to determine the formation constants for the complexation equilibria. Accordingly CD could be used in the determination of achiral compounds ((0. Examples of induced CD activity were previously reported from this laboratory using first a cholesteric liquid crystalline solvent (9),which could not be exploited for quantitative studies, and second for L-cocaine and phencyclidine (PCP) using @-cyclodextrin(10). In this work the complexation with PCP has been reexamined, and seven other inherently achiral drug molecules are included for comparison.
EXPERIMENTAL SECTION P-Cyclodextrinwas obtained from Eastman Kodak and used without further purification. The eight guest molecules were PCP and the pyrrolidine (PCPy) and morpholine (PCM) hydrochloride analogues (Applied Science), P-phenethylamineand phenobarbital (Sigma Chemical Co.), meperidine hydrochloride (Sterling-Winthrop), and diazepam and dilantin (Drug Enforcement Administration). Solution concentrations of the guest compounds ranged from 5X M to be consistent with a solution absorbance to which is within the dynamic range of the CD instrument. The first five compounds were dissolved in distilled water. Diazepam was dissolved in 0.1 M hydrochloric acid and dilantin and meperidine in 0.1 M sodium hydroxide because of their limited solubilities in water. @-Cyclodextrinis stable in dilute base but is
0003-2700/84/0356-2822$01.50/00 1984 American Chemlcal Society
ANALYTICAL CHEMISTRY, VOL. 56, NO. 14, DECEMBER 1984
I
A
t
+
2823
I
B
U
I
I
0-
CH
N
U
I
E E
7
+
-
x
220
320
G
F H3
+
- 220
H x 320
Flgure 1. Induced CD spectra for (A) PCP, (6)PCPy, (C) PCM, (D) P-phenethylamine,(E) phenobarbital, (F) meperidine, (G) diazepam, and (H) dilantin. The wavelength scale is the same for all. The ordinate is the ellipticity in arbitrary units. (Experimental data available on request.)
rapidly hydrolyzed in acid solutions which are greater than 0.1 M concentration. For the calculations of the formation constants spectra were obtained for a series of solutions in which aliquots of a stock 0-cyclodextrin were added to a fixed concentration of guest up M. Wherever possible to a maximum host concentration of 0-cyclodextrin was in molar excess. Additions were made by volume if sufficient quantities of guest were available to allow the performance of replicate experiments and by weight where supplies were limited. CD measurements were made an a JASCO-500A automatic recording spectropolarimeter with data collection and analyses made on the ancillary DP-BOON data processor. Daily calibration of the ellipticity scale was made against a standard solution of
androsterone in dioxane as recommended. Measurements were made over the wavelength ranges of the absorptions attributed to the aromatic ring and carbonyl chromophores. Sensitivity,scan rate, and repeat functions were selected which optimized the signal-to-noise ratio.
RESULTS AND DISCUSSION The induced CD spectra for the eight guests are shown in
-
Figure 1. With three exceptions the three a* a aromatic transitions at wavelengths between 250 and 275 nm are well represented. The chirality of these bands is principally negative. There are different and statistically reproducible ratios for the peak maxima from compound to compound
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ANALYTICAL CHEMISTRY, VOL. 56, NO. 14, DECEMBER 1984
Table I. Formation Constants and Induced Molar Ellipticites for Drug-Sugar 1:l Complexes
compound
K"
PCP PCPy
1024 712
PCM
460
P-phenethylamine meperidine phenobarbital
-0.9
-1.6 -1.8 +0.3 -1.6 -2.0 -0.9
82 134 1098
diazepam
-1.9
+12.5 +16.0 +12.8 -25.1 +151.8 -426.7
83
dilantin a
ODsb
144
A, nm
271 27 1 264 235 271 265 270 271 273 268 350 307 260 235
+
SD is 120. * SD is f0.3.
which, together with the variability i n l h e sign of the 240-nm band, allow for a qualitative distinction among the five analytes. The spectrum for dilantin is the first exception to this pattern. For this compound a single negative Cotton band is observed with a maximum a t 235 nm. Both it and meperidine were complexed with the host in dilute base, but only the spectrum of meperidine shows the typical aromatic transitions. The apparent absence of these bands for dilantin is not necessarily attributable to the alkaline conditions because the bands persist for alkaline solutions of phenobarbital and P-phenethylamine as well as for meperidine. It must be assumed that either the rotational strengths for these transitions in dilantin are lower than the detection limit of the instrument at the limiting concentrations used or the bands are concealed under the major band observed. The other two exceptions to the general pattern are phenobarbital and diazepam. All three exceptions contain a carbonyl chromophore whose CD activity dominates the spectrum at wavelengths longer than 250 nm. A positive band is induced on complexation for phenobarbital, while both positive and negative bands are evident for diazepam. Characteristic spectral parameters are given in Table I. When the molecular interaction between drug (D) and sugar (S) is treated as a 1:1complexation equilibrium, molar concentrations are related to the experimental ellipticities, +, by the equation $ = oD[Dl + ODS[DS]
(1)
where OD and ODs are the molar ellipticity coefficients for the free and complexed drug. OD is of course zero for achiral molecules and eq 1 reduces to = 6 ~ s [ D s ] so , that the equation for K , the formation constant, becomes
+
where CD and Cs are total analytical concentrations. K and ODs were simultaneously evaluated in a convergent iterative routine using an equation analogous to one from the older literature for visible-UV absorption data ( I I ) , namely
Wl IS1
-= ($ - rc/D)
ID1
+ [SI - [DSI (ODs
- OD)
+
1 K(8DS - OD)
intensity. Results are given in Table I. K values ( k 2 0 ) are overall averages. ODS values (f0.3) correspond with the wavelengths given in parentheses. The weakest interaction is observed between P-cyclodextrin and P-phenethylamine in terms of both the K value and the induced molar ellipticity. The value of 8 ~ iss similar to that calculated for DL-a-phenethylamine and almost one tenth of - L-a-phenethylamine alone in the 8, value for either ~ - aor aqueous solution. P-Phenethylamine might be representative, therefore, of the minimum affect of complexation on a CD spectrum assuming the side chain has no influence on the interaction. All other effects are much greater, but the magnitude of is determined by the product of ODs[DS]and is therefore a consequence of either K or ODs (or both), being comparatively large. For the PCP derivatives and meperidine the ODs(271 nm) values are very comparable which is not unexpected because of the strong structural resemblances. The reason for the range in K is not apparent. A previous K value for phencyclidine hydrochloride (IO) was reported as very uncertain. This is in part due to the lower sensitivity of the older Cary 61 (CD) instrument used in that work and in part due to the mathematical procedure employed which used a graphical convergence criterion. Data from the study for L-cocaine were reanalyzed using the present procedure. A good correspondence in K was obtained, giving us more confidence in the present results for achiral substances. Diazepam, dilantin, and phenobarbital contain one, two, and three alicyclic carbonyl chromophores, respectively. No spectral changes are observed which are consistent with this trend. The presence of a carbonyl group does appear to affect s maxima around 270 nm. the sign and magnitude of 8 ~ for For the most part the CO groups are a t least two carbons removed from the aromatic ring. If the structure of the guest external to the interaction site does not contribute toward the stability of the complex (1-3)) then the observed trend in K must be coincidental. The largest ODsfor a particular chromophore would be indicative of the greatest susceptibility to chirality induction. In this limited group ODs is greatest for diazepam, the value for dilantin describing a different electronic transition. Of the eight compounds investigated, diazepam has the most structural rigidity. In analytical applications the problem of interference from other mixture components is very real. If P-cyclodextrin binds every component with an aromatic chromophore indiscriminately, the technique is severely limited. The eight analytes described here are all monosubstituted benzene derivatives, and their simultaneous occurrence would require prior separation. In contrast, in a preliminary study of a few compounds in which the aromatic ring is substituted more than once, e.g., quinine, aspirin, mescaline, and psilocin, there is no evidence for CD induction on complexation, if there is indeed complexation. If this proves to be more general, then aromatic compounds of this type would be noninterfering. In the following paper in this issue there is evidence for inclusion of nonaromatic structural moieties under particular solution conditions (12). Commercially available Demerol tablets (Winthrop) were directly assayed for meperidine (50 mg) using the induced CD spectral data obtained for a simple solution of the tablet in a standard alkaline P-cyclodextrin ( M) stock reagent. Correspondence with the prescribed amount was better than 98%. The technique might be suitably developed for quality control applications in the pharmaceutical industry.
(3)
where b is the cell path length in centimeters. Iteration is initiated by assuming [DS] = 0, and the values of ODs and K obtained from the slope and intercept of eq 3 are refined by substituting new values for [DS] until successive K values differ by unity. Determinations were done in replicate, and calculations were made at all wavelengths of maximum signal
ACKNOWLEDGMENT We are indebted to the Dallas office of DEA for their assistance in obtaining the samples.
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Anal. Chem. 1984, 56, 2825-2827
LITERATURE CITED (1) Bender, M. L.;Domiyama, M. "Cyclodextrln Chemistry"; Springer-Verlag: New York, 1978. (2) Saenger, V. W. Angew. Chem. 1980, 9 2 , 343. (3) Frank, S. G. J . fharm. Sci. 1975, 6 4 , 1585. (4) Smoikova-Keulemansova, E. Chromatogr. Rev. 1982, 257, 17. (5) Harata, K. Bull. Chem. SOC.Jpn. 1978, 51, 2737. (6) Gelb, R. I.; Schwartz, L. M.; Cardelino, B.; Fulerman, H. s.; Johnson, R. F.; Lanfer, D. A. J. Am. Chem. SOC. 1981, 103, 1750. (7) Schipper, P. E.; Rodger, A. J. Am. Chem. SOC. 1983, 105, 4541. (8) Thakkar, A. L.; Kuehn, P. B.; Perrin, J. H.; Wllham, W. L. J . fharm. Sci. 1972, 6 1 , 1841.
(9) Bowen, J. M.; Crone, T. A.; Hermann, A. 1980, 5 2 , 2436.
0.;Purdie, N. Anal. Chem.
(IO) Bowen, J. M.; Purdie, N. Anal. Chem. 1981, 5 3 , 2239. (1 1) Monk, C. B. "Electrolytic Dlssociation"; Academic Press: New York, 1961; p 186. (12) Han, S. M.; Purdie, N. Anal. Cttem., following paper in this issue.
RECEIVED for review April 13,1984. Resubmitted August 9, 1984. Accepted August 27,1984. Support of this work was from the Science Foundation under Grant NSF CHE-8240564.
Cyclodextrin Complexation of Barbiturates in Aqueous Solution Soon M. Han and Neil Purdie* Chemistry Department, Oklahoma State University, Stillwater, Oklahoma 74078
Clrcular dlchrolsm spectropolarlmetry has been used to determlne the formatlon constants for the 1:l complexes obtalned between @-cyclodexlrlnand elght barbltals. Data for secobarbltal have been used to assay commerclal seconal sodium supposltorles wlthout prlor mlxture separatlon. Correlations are made between the values of the formation constants and the structural varlatlons among the analogues. Thermodynamic constants are reported for the three most closely related analogues. The evldence suggests that the allphatlc substltuents are Included In the cyclodextrln core, a phenomenon which could be explotted In the separatlon of a mixture of aliphatic anions.
In previous work, (1)the application of circular dichroism (CD) spectropolarimetry to the study and determination of achiral substances has been described. This is accomplished by first complexing the achiral molecule with a chiral host, namely fl-cyclodextrin (BCD), calculating the equilibrium constant for the association from the concentration dependence of the induced CD spectra, and using this value for the direct determination of the analyte in a mixture. Separation of the analyte from the mixture is not always necessary. In this work the complexation constants for eight barbiturate analogues have been calculated and determinations have been made on a commercially available product. Trends in the formation constants and enthalpies and entropies of complexation and in the induced molar ellipticities with structural modifications of the molecules are discussed. Results are compared with values from previous studies (1,2).
EXPERIMENTAL SECTION BCD was obtained from Eastman Kodak and used without purification. Barbituric acid and the seven 5,5-disubstituted analogues, allo-, amo-, buta-, hexa-, pento-, pheno-, and secobarbital were Sigma Chemical Co. products. The 5,5-diethyl derivative (barbital) available was provided by Mallinckrodt, Inc. Seconal sodium suppositories (Eli Lilly) were supplied by a local pharmacy. All CD measurements were made with the analytes and BCD dissolved in aqueous 9.8 buffer medium (pHydroin). The barbital concentrations used ranged from 0.5 to 2.0 x M which produced a substantial induced CD signal at wavelengths longer than 250 nm. Below 250 nm the signal quality deteriorated rapidly due to excessive absorption. For the calculation of formation constants solid BCD was added incrementally to a stock solution of the analyte up to a maximum concentration of low2
M, so that BCD was kept always in excess. The temperature dependences of the formation constants were calculated for amo-, buta-, and pentobarbital from duplicate data obtained at 15,25, and 35 "C, respectively. Temperatures were controlled to *0.1 "C using water circulated around the cuvette from an external thermostat (Haake, Model A81). CD measurements were made on a JASCO-500A, DP-BOON automatic recording spectropolarimeter-data processor assembly. Daily calibration of the ellipticity scale was made against a standard solution of androsterone in dioxane as recommended. Sensitivity, scan rate, and repeat functions were selected which optimized the signal-to-noise ratio.
RESULTS AND DISCUSSION The general structural formula for the barbital analogues is represented by Figure 1. All are 5,5-disubstituted derivatives of barbituric acid, and only hexabarbital has further substitution, namely a methyl group on the nitrogen in position 1. pK1 values for all except barbituric acid are in the range 7-8.5 (3), so the only species in pH 9.8 buffer is the enolized monoanion. The pK value for the ionization of the second proton is in excess of 13 (3). Induced CD spectra are shown in Figure 2 for secobarbital and phenobarbital. The spectrum for secobarbital is typical of the spectra for all the compounds except phenobarbital which, as would be expected ( 4 ) ,shows the familiar triplet of aromatic ring a* a or lLb transitions. With the exception of phenoharbital the method is incapable of distinguishing among the various derivatives when each occurs separately, but recognition of an anonymous analyte as a barbiturate is possible. No CD spectrum is obtained for barbituric acid either under these experimental conditions or in acid media. When the induced CD spectra and the changes are treated with increasing concentrations of BCD as evidence for a 1:l drug-sugar (DS) complex, the experimental ellipticity, $, measured is equal to $~s[Ds] where I ~ isD the ~ molar-induced ellipticity coefficient and [DS] is the equilibrium concentration of complex (I). In order to calculate the formation constant K, ODS must first be approximated and subsequently refined in an iterative manner which terminates when both K and ODs are invariant. This is conveniently done by the solution of the general equation for absorbance measurements (5) modified to accommodate ellipticities
-
where b is the sample path length in centimeters, $D and OD
0003-2700/84/0356-2825$01.50/0 0 1984 American Chemical Society
