Anal. Chem. 1989, 6 1 , 1171-1174
in the high-pH regions shown in Figure 3 are ascribable to an interference from hydroxide. In a previous paper (12),we reported selectivity sequences for the Co(II1)-TPP anion carrier and a classical anion exchanger (trioctylmethylammonium chloride). By comparing reported selectivity coefficients of both types of solvent polymeric membranes which have almost the same membrane matrices as those in the present work, one can roughly estimate selectivity changes of thiocyanate and nitrite induced by the Co(II1)-TPP anion carrier. Since perchlorate does not readily complex with the metal center, this is selected as a reference ion in estimations of selectivity changes. For the classical anion-exchange solvent polymeric membrane, log kC104-,N02and log kC104-,SCN- are -4.6 and -1.3, respectively. In the case of the Co(II1)-TPP anion carrier based solvent polymeric membrane, log kC104-,N02- is 1.6 and log kC104-,SCN- is 2.7. Thus, selectivity changes for nitrite and thiocyanate induced by the Co(II1)-TPP anion carrier relative to those of the classical exchanger are estimated to be 6 and 4 orders of magnitude, respectively. Although detailed studies on associations of the complex cation with nitrite as well as with thiocyanate are required, it can be estimated that nitrite associates with the carrier more strongly than thiocyanate does. Consequently, complexes in the nitrite form cannot contribute as much to overall conductivities of solvent polymeric memrbanes as complexes in the thiocyanate form do. This suggests that the blank response of the membrane matrix will be reflected more strongly in the case of complexes in the nitrite form than in the case of complexes in the thiocyanate form. Indeed, solvent polymeric membranes containing the Co(II1)-TPP in the thiocyanate form showed virtually no pH dependence in response to thiocyanate (12),while ones containing complexes in the nitrite form showed the strong pH dependence in response to nitrite, as is shown in Figure 1. In contrast to the potentiometrically active plasticized PVC matrix, the anomalous pH effect shown in Figure 1 is successfully eliminated in the potentiometrically inert liquid membrane matrix (Figure 3). These results imply that the anomalous pH effect observed in responses of solvent polymeric membranes containing complexes in the nitrite form comes from the membrane matrix, although the whole mechanism of this pH-dependent response is not now clarified. As is shown in this work, there is a potential for unfavorable
1171
side effects from membrane matrices in responses of positively charged anion carrier based solvent polymeric membrane electrodes when anion carriers having a monovalent positive charge, like the presented complexes, interact too strongly with a specific anion like nitrite.
LITERATURE CITED (1) Schulthess, P.; Ammann, D.; Krautler, B.; Caderas, C.; Steplnek, R.; Simon, W. Anal. Chem. 1985, 5 7 , 1397-1401. (2) Ammann, D.; Huser, M.; Krautler, 8.; Rusterholz, B.; Schulthess, P.; Lindemann, B.; Hadler, E.; Simon, W. Helv. Chim. Acta 1988, 6 9 , 849-854. (3) Chaniotakis, N. A.; Chasser, A. M.; Meyerhoff, M. E.; Groves, J. T. Anal. Chem. 1988, 6 0 , 185-188. (4) Horvai, G.; Grlf, E.; T6th, K.; Pungor, E.; Buck, R. P. Anal. Chem. 1986, 58, 2735-2740. (5) T6th. K.; Grlf. E.; Horvai, G.; Pungor. E.: Buck, R. P. Anal. Chem. 1988, 5 8 , 2741-2744. (6) Van den Berg, A.; van der Wal, P. D.; Skowrofiska-Rasiirska, M.; Sudholter, E. J. R.; Reinhoudt, D. N.; Bergveld, P. Anal. Chem. 1987, 5 9 , 2827-2829. (7) Sugimoto, H.; Tooda, K.; Suzuki, K.; Shirai, T. Abstr. No. 2IVB 01, 54th National Meeting of the Chemical Society of Japan, Tokyo, April 1987. (8) Masadome, T.; Imato, T.: Ishibashi, N. Anal. Sci. 1987, 3, 121-124. (9) Allen, C. F. H.; Gates, J. W., Jr. Organic Synthesis Collective Volume 3; Hornig. E. C., Ed.; John Wiley & Sons, Inc.: New York, 1955; pp 140, 141. (10) Adler, A. D.; Longo, F. R.; Kampas. F.; Kim, J. J . Inorg , Nucl. Chem , 1970, 32, 2443-2445. (11) Sakurai, T.; Yamamoto, K.; Naffo, H.; Nakamoto, N. Bull. Chem. SOC. Jpn. 1978, 4 9 , 3042-3046. (12) Hodlnar, A.; Jyo, A. Chem. Len. 1988, 993-996. (13) Anker, P.; Wieland, E.; Ammann, D.; Dohner, R. E.; Asper, R.; Simon, W. Anal. Chem. 1981, 5 3 , 1970-1974.
Ale6 HodinHi. Laboratory for Endocrinology and Metabolism Faculty of Medicine Charles University at Prague U Nemocnice 1 Prague, CSSR Akinori Jyo* Department of Applied Chemistry Faculty of Engineering Kumamoto University 2-39-1 Kurokami Kumamoto 860, Japan RECEIVED for review March 23,1988. Accepted February 24, 1989.
Effect of Protein Binding on the High-Performance Liquid Chromatography of Phenytoin and Imirestat in Human Serum by Direct Injection onto Internal Surface Reversed-Phase Columns Sir: Internal surface reversed-phase (ISRP) columns have been designed to facilitate the HPLC analysis of drugs in blood serum or plasma by direct injection (1). The ISRP concept consists of binding a diol-gly-phe-phe peptide partitioning phase to the internal surface of 5 pm porous silica, while rendering the external surface hydrophilic and nonabsorptive to proteins via a glycerylpropyl bonded phase. The peptide-bonded phase is removed from the external surface of the supports by enzyme cleavage (2). The final median pore diameter of the packing is 52 A, so serum proteins are size excluded from the internal regions of the packing (3). Drugs with low molecular weights (CZOOO) penetrate the ISRP packing and partition with the internal peptide-bonded phase.
Serum proteins, on the other hand, elute in the column interstitial void. The diol-gly-phe-phe internal bonded phase favors the retention of aromatic drugs and separates analytes primarily by a reversed-phase mechanism ( 4 ) . With the carboxylic acid terminal on the peptide-bonded phase, the packing exhibits a secondary cation-exchange mechanism and provides strong selective control for positively charged aromatic amines on variation of mobile phase ionic strength ( 5 ) . The ISRP columns have been used for the direct high-performance separation of a wide variety of drugs in serum or plasma (4-7); for the precolumn switching isolation of substances from serum or plasma ( 1 , 8, 9); for the determination endogenous me-
0003-2700/89/0361-1171$01.50/00 1989 American Chemical Society
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ANALYTICAL CHEMISTRY, VOL. 61, NO. 10, MAY 15, 1989
H
A
I H Figure 1. Chemical structures of (A) phenytoin and (B) imirestat.
tabolites in uremic serum, such as indoxyl sulfate (10,11); for the separation of biologically active peptides (12);and for the assay of peptide toxins from cyanobacteria (13). The development and utilization of ISRP columns have been recently reviewed (14). The following work describes a unique split-peak phenomenon which can occur when some strongly protein bound substances, such as phenytoin and imirestat in serum, are directly injected onto an ISRP columns under carefully controlled conditions. The resulting two peaks enable one to estimate the degree of protein binding. EXPERIMENTAL SECTION Instrumentation. The chromatographic system consisted of a Milton Roy Model 396 pump (LDC, Riviera Beach, FL), a Rheodyne 7010 six-port valve with pneumatic actuator, and a Beckman Model 153 UV-visible detector equipped with a 254-nm filter. A Hewlett-Packard 3390A integrator was used to determine peak areas. Automated injection was facilitated by a sample loop which could be filled with a FMI Model RHSYOCKC pump (Fluid Metering, Inc., Oyster Bay, NY) via Teflon tubing. Both the FMI pump and the injector actuator were controlled by a Valco Model DVSP-4 digital sequence programmer. The columns used during the study were 5 cm x 4.6 mm i.d. packed with 5-pm ISRP supports (GFF-S5-80)obtained from the Regis Chemical Co., Morton Grove, IL. Except where stated otherwise, a serum sample size of 200 pL was employed. The stainless steel inlet frit was replaced by a titanium/Teflon frit/filter arrangement described elsewhere ( 4 ) . Reagents and Standards. Water used in the study was deionized, glass distilled, and purified to 18 MQ with a Gelman Water I unit. Buffer solutions were made from HPLC Grade potassium dihydrogen phosphate (Fisher Scientific)and adjusted to the appropriate pH with sodium hydroxide. Sodium azide (0.05% by weight) was added to the buffers to act as a preservative. The solutions were filtered through 0.2-pm filters and vacuum degassed prior to use. Phenytoin (99+% pure) was obtained from Aldrich Chemical Co., Milwaukee, WI. The imirestat (spiro[2,7-difluorofluorene]-9,4’-imidazolidine-2’,5’-dione, AL1576,98+% pure) was generously donated by Alcon Laboratories, Fort Worth, TX. Stock solutions of each drug were prepared in methanol. Pooled human serum was obtained from Pel-Freez Biologicals (Brown Deer, WI). Serum was stored frozen and thawed before use. Aliquots of serum were filtered through 0.2-pm nylon filters under gentle vacuum prior to the addition of drug. Spiked serum samples were prepared from methanol stock solutions in such a manner that the methanol never exceeded 0.5%. Once prepared, spiked serum samples were allow the equilibrate for 30 rnin with gentle shaking. R E S U L T S A N D DISCUSSION Discovery of Split-Peak Phenomenon. Phenytoin and imirestat (Figure 1) are strongly retained on ISRP columns under conditions established for the analysis of drugs in serum or plasma by direct injection ( I , 4 , 15). Typically, a 15-cm ISRP columns are employed along with 10-pL injections and
m
R 0 20
0 10 20 TIME ( m i d
Figure 2. Split-peak phenomenon observed from the direct injection of (A) 200 WLof imirestat (20 pg/mL) in human serum and (B) 500 WL ot phenytoin (51 pglmL) in human serum, onto a 5-cm ISRP column: mobile phase, 0.01 M phosphate (pH 6.8); flow rate, 1.0 mLlmin; detection, 254 nm with 0.08 AUFS.
mobile phases consisting of 80% 0.1 M phosphate buffer/20% acetonitrile or 84% 0.1 M phosphate buffer/lO% 2propanol/6% tetrahydrofuran ( 4 ) . Although ultrafiltration studies indicate that phenytoin is 93% bound to serum proteins at therapeutic concentrations ( I 6 ) ,when serum is directly injected onto ISRP columns the drug elutes as a single peak with recoveries ranging from 97 to 100% ( I ) . Under these moderately strong (yet nondenaturing) mobile phase conditions, the drug dissociates from the proteins, and partitions with the internal chromatographic phase. In an attempt to chromatographically preconcentrate the imirestat and achieve lower limits of detection, serum samples in excess of 200 pL were injected onto a 5-cm ISRP column using only an aqueous buffer as a mobile phase. Unexpectedly, rather than simply extracting onto the bonded phase, the imirestat eluted as two peaks, after the serum protein peak (Figure 2A). The same phenomenon was observed with phenytoin (Figure 2B). The injection of each drug without serum yielded retention times corresponding t o the second peaks. The injection of serum alone ruled out serum artifacts, and the use of six ISRP columns demonstrated that the effect was not unique to one column. The ultraviolet-visible spectrum for each peak was determined with on-line photodiode array detection and found to be identical. Quantitatively, the two peaks accounted for 100% of the drug injected. The small peak represented approximately 7.5% of the total drug. Since the phenytoin was 99+% pure and imirestat was 98+% pure, it was surmised that the peaks were fractional parts of the drug, which had been split by an on-column effect. The observation of this phenomenon was first reported a t the 30th Annual Symposium on Liquid Chromatography in Japan (17).
Variation in Sample. At a constant mobile phase buffer concentration of 0.01 M phosphate (pH 6.8), separations were conducted on a 5-cm ISRP column with variation in sample volume of serum spiked with drug. As the sample size increased from 55 to 660 wL,the retention time of the first drug peak decreased, while the retention time of the second peak remained constant (Figure 3). This indicated the split-peak phenomenon was uniquely dependent on sample size. In order to test whether or not the split-peak phenomenon was a function of interactions with the serum sample matrix
ANALYTICAL CHEMISTRY, VOL. 61, NO. 10, MAY 15, 1989
1173
1
t
'9 0
.-
\
I\.
22
k
18
i
-I
P c
Figure 3. Effect on split-peak of sample size with the Chromatographic elution of 20 pglmL of imirestat in human serum from a 5-cm ISRP column. Conditions were same as Figure 2 with detector attenuations of 0.04, 0.08, 0.16, 0.32, and 0.64 for 55-660 pL, respectively. Numbers over peaks are retention times in minutes.
10
I 0
I
200
400
600
800
SALICYLIC ACID CONC oJg/mL)
Protein binding displacement of imirestat by salicylic acid. The amount of free drug was determined by the area of the second peak. Conditions are the same as those given in Figure 2. Figure 4.
or simply sample size, a 50 pg/mL phenytoin standard in a 0.5% methanol solution of 0.1 M or 0.01 M phosphate buffer was injected over a range of sample sizes up to 1 mL. In all cases, only one peak was observed. The same was found to be true with imirestat. The injection of buffer blanks eliminated the possibility of background artifacts. This indicated that the split-peak phenomenon was not caused by a column overloading and that the presence of a serum matrix was required. In order to evaluate whether the sample matrix interactions were a function of small endogenous serum components or the protein fraction, serum was subjected to ultrafiltration, as described elsewhere (15), and drug-spiked serum ultrafiltrate was injected. Only one drug peak appeared, regardless of sample size or mobile phase conditions. This led to the conclusion that the split-peak phenomenon necessitated the on-column presence of drug and serum proteins. If the two peaks result from protein binding, it is logical that the addition of a protein binding displacer to the sample would perturb the effect. To test this hypothesis salicylic acid, over a concentration range from 200 to 800 pg/mL, was added to drug spiked serum. Salicylic acid can bind with the warfarin site but is not well retained on the 5-cm ISRP column under these conditions and does not interfere with the drug peaks. As can be seen from Figure 4, when the displacer concentration increases, the area of the second peak increases relative to the first, thus exhibiting a higher free drug concentration. Variation i n Mobile Phase. The retention of the phenytoin and the split-peak phenomenon are dependent on a mobile phase containing buffer. Varying the mobile phase phosphate buffer concentration from 0.0025 to 0.015 M, at a constant pH of 7.4, generated a change in the relative retention of the split peaks. As the mobile phase ionic strength decreases, the retention of the first peak decreases, while the retention of the second peak remains constant. At constant ionic strength and sample size, the variation in mobile phase pH from 6.5 to 7.4 decreases the retention of both peaks, increases the ratio of retention times from 1.15 to 1.28, and increases the peak height of the first peak at the expense of the second. The inclusion of 2-3% of acetonitrile in the mobile phase decreased the overall retention of .both peaks, improved peak shape, and increased relative retention; however, with a 5-cm ISRP column the peaks became more difficult to resolve from the serum protein peak (15). D r u g Protein Binding. The splitting of the chromatographic drug fraction into two peaks is facilitated when the following conditions are met: the sample contains binding proteins, the sample size is 1200 pL, and the mobile phase is weak. Because the peak splitting occurs only with the presence of protein and the peak height ratios change as a function of the increasing protein binding displacer, one can
conclude the phenomenon is a result of drug protein binding. In view of this, it is instructive to briefly comment on the protein binding of drugs in serum. There are primarily three types of proteins in blood serum which bind drugs. These include albumin, which binds neutral and acidic drugs; a acid glycoproteins, which bind neutral and basic drugs; and lipoproteins, which bind very hydrophobic drugs (18). Albumin is the most abundant and the most pervasive in drug binding. I t is known to have five binding sites, three of which bind small organic molecules. These sites are classified according to their binding displacers, which are the warfarin site, the diazepam site, and the digitoxin site. It has been commonly accepted that the primary binding of phenytoin occurs at the warfarin site, becuase of displacement by such species as salicylic acid (19). However, considerable controversy exists regarding the true location of phenytoin's secondary binding site. Some believe that both binding sites are on albumin (19, 20), while others speculate that the secondary site is located on another protein (21). Quantitatively, the moles of drug bound per mole of total protein ( r ) can be expressed by
nlKID r = 1+KID
nzKzD
+ 1 + KzD
(1)
where D is the free drug concentration, ni is the number of drug molecules bound to each type of binding site, and K iis the affinity constants for each site (22). Separation Mechanism. The split-peak phenomenon appears to be governed by the rate a t which the drug is released from the binding proteins on introduction to the chromatographic mobile phase. As the serum sample is injected into the ISRP column, the proteins are excluded from the packing intraparticulate space (due to the small pore diameter) and confined to the interstitial volume. Studies have shown that the ratio of pore volume to interstitial volume for ISRP packing is 0.5 (3). With a total void volume of 600 pL for a 5-cm column, the interstitial volume would be 400 pL and the pore volume 200 pL. With a 200-pL serum sample occupying half of the column interstitial volume, it becomes clear that the protein concentration in the sample would remain essentially constant with dilution only at the beginning and end of the sample bolus. On the other hand, small molecules (Le., drug, buffer ions, etc.) will exchange with the stagnant mobile phase in the intraparticulate space and undergo dilution throughout the sample. In like manner solvent molecules,ions, and organic modifier (if present) would diffuse into the sample bolus from the stagnant mobile phase. One can presume that the free drug in the sample readily diffuses
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ANALYTICAL CHEMISTRY, VOL. 61, NO. 10, MAY 15, 1989
into the packing and partitions with the internal phase. As the concentration of the free drug in the interstitial volume of the sample bolus decreases, the protein binding equilibrium is upset and the amount of drug bound decreases rapidly (eq l),thus driving the dissociation during the sample loading process. For a 200-pL sample at a flow rate of 1.0 mL/min the sample loading process would take 12 s. Obviously, the size-excluded, nonpartitioning protein-bound drug complex moves down the column with a more rapid migrational velocity than the drug alone, thus providing the necessary separation immediately prior to releasing the drug during the sample loading process. Consequently, the first eluting peak represents the drug previously bound to protein, while the second peak represents the free drug or any drug released immediately on sample introduction. The larger the sample, the longer the resident time of the loading process, and the lower the overall dilution factor. This results in a slower release of the drug and an earlier elution of the first peak (Figure 3). Although dilution can be viewed as the primary driving force behind the protein-binding drug dissociation, one cannot overlook the importance of kinetic effects. A change in the rate of dissociation during the sample loading process can occur from changes in ionic strength, pH, or organic solvent brought about by diffusion of species from the stagnant mobile phase into the sample bolus. In the absence of such effects one might expect a slower release of drug, which would result in one broad band. With strongly bound drugs, such as warfarin, this is in deed the case, and the presence of a small percentage of organic modifier in the mobile phase accelerates the release (23). From the above discussion one can conclude that the area of the first peak should approximate the drug bound to the protein prior to injection and that the second peak should represent the free drug and any drug released immediately on introduction to the column. At a therapeutic phenytoin concentration of 20 pg/mL, the fraction of drug bound to the serum protein was determined to be 9370, based on the areas of the two peaks and the assumption that the second peak represented only free drug. This is consistent with results determined by other methods, such as ultrafiltration (16,24). It has not been determined whether the fraction bound by this ISRP method would be consistent with other methods over a wide concentration range. In the original work, an attempt was made to evaluate the phenytoin binding at higher drug concentration by the ISRP method; however, complications arising from peak overlap and inadequate peak integration methods precluded interpretation (15). Subsequent work by investigators at Kyoto University (23) using warfarin, bovine serum albumin (BSA), a 4.6 mm X 25 cm ISRP column, and a 200-pL sample size has demonstrated the same type of peak-splitting phenomenon. In this case, it was necessary to use 5% tetrahydrofuran in the mobile phase to facilitate a release of the warfarin from the BSA. The amount of free drug appearing at the second peak was higher than that expected compared to ultrafiltration results on the same samples. It was postulated that the amount of drug in the second peak was due to drug previously bound to secondary binding sites as well as free drug. Since the type and concentration of the protein were known, determination of
the amount bound to the secondary site by difference enabled a direct calculation of binding constants for each type of binding site (eq l ) ,yielding log K1 = 4.87 0.31 and log K 2 = 3.54 f 0.19, assuming n, = 2 and n2 = 5 . These values compared well with previous determination using equilibrium dialysis, which gave log K , = 5.06 and log K 2 = 3.21 (2i3).
*
ACKNOWLEDGMENT The authors wish to thank Alcon Laboratories, Inc., Fort Worth, TX, for generously supplying imirestat. LITERATURE CITED Hagestam, I . H.; Pinkerton, T. C. Anal. Chem. 1985, 57, 1757-1763. Pinkerton, T. C.; Hagestam, I.H. US. Patent 4,544,485,Oct 1985. Cook, S. E.; Pinkerton, T. C. J. Chromatogr. 1988, 368,233-248. Pinkerton, T. C.; Miller, T. D.; Cook, S. E.; Perry, J. A.; Rateike, J. D.; Szcerba, T. J. BioChromatography 1988, 1(2),96-105. Nakagawa, T.; Shibukawa, A.; Shimono, N.; Kawashima, T.; Tanaka. H.; Haginaka, J. J. Chromatogr. 1987, 420, 297-311. Pinkerton. T. C.;Perry, J. A.; Rateike, J. D. J. Chromatogr. 1988,
367,412-418. Perry, J. A.; Rateike, J. D.; Szczerba, T. J. Application Notes; Regis Chemical Company: Morton Grove, IL. Chu, Y.4.; Wainer, I. W. Pharm Res. 1988, 5(10), 680-683. Mathes, L. E.; Muschik, G.; Demby, L.; Polas, P.; Mellini, D. W.; Issaq, H. I.; Sams, R. J. Chromatogr. 1988, 432, 346-351. Niwa. T.; Takeda, N.; Tatematsu. A,; Maeda, K. Clin. Chem. 1988,
34(11).2264-2267. Takeda, N.; Niwa. T.; Maeda, K.; Shibata, M. J. Chromatogr. 1988,
43 1 418-423. Pinkerton. T. C.; Koeplinger, K. A. J . Chromatogr. 1988, 458,
129-145. Meriluoto, J. A. 0.; Eriksson, J. E. J. Chromatogr. 1988. 438,93-99. Pinkerton, T. C. Am. Lab. 1988, 20(4),70-76. Miller, T. D. Ph.D. Thesis, Purdue University, West Lafayette, IN, August 1987. Miller, T. D.; Pinkerton, T. C. Anal. Chim. Acta 1985, 170,295-300. Pinkerton. T. C.; Cook, S.E.; Desilets, C. P.; Miller, T. D. “Analysis of Drugs in Serum by Direct InJection with Internal Surface ReversedPhase Chromatography”; Plenary lecture presented to the 30th Annual Symposium on Liquid Chromatography in Japan, Kyoto, Japan, January 27-28, 1987. Marcus, M.: Reidenberg, M. D.: Sergio, E. Drug-Protein Binding: Praeger: New York. 1986. Sjoholm, I.; Ekman, 6.; Kober, A.; Ljungstedt-Pahlman, I.; Selving, B.; Sjodin, T. Mol. Pharmacol. 1979, 16, 767-777. Arrons, L. J.; Schary. W. L.; Rowland, M. J. pharm. Pharmacol. 1979,
37, 322-330. Odar-Ceder, I.; Borga, 0. Clin. Pharmacol. Ther. 1978, 20,36. Meyer, M. C.; Gunman, D. E. J. Pharm. Sci. 1988, 57(6),895-918. Shibukawa, A.; Nakagawa, 1.;Miyake. M.; Tanaka, H. Chem. Pharm, Bull. 1988, 36(5),1930-1933. Lunde, K. M.; Rane, A.; Yaffe, S.J.; Lund, L.; Sjoqvist, F. Ciin. Pharmacoi. Ther. 1970, 11(6),846-855. Brown, K. F.; Crooks, M. J. Biochern. Pharmacol. 1976, 1175.
‘
Current address: Control Division, Bldg 259-12,The UpJohn Company, Kalamazoo. M I 49001. ‘Current address: Marion Laboratories, Inc., Park B - P.O. Box 9627, Kansas City, MO 64134.
Thomas C. Pinkerton*J Terry D. Miller2 Linda J. Janis Department of Chemistry Purdue University West Lafayette, Indiana 47907 RECEIVED for review August 8, 1988. Resubmitted February 7 , 1989. Accepted February 28,1989. This investigation was supported by Public Health Service Grant R01-GM34759-01 awarded by the National Institutes of General Medical Sciences, Department of Health and Human Services.
