Separation of Hydrophobic, Positively Chargeable Substances by

Separation of Hydrophobic, Positively Chargeable Substances by Capillary Electrophoresis. Chao-Xuan. Zhang, Franz. von Heeren, and Wolfgang. Thormann...
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Anal. Chem. 1995, 67,2070-2077

Separation of Hydrophobic, Positively Chargeable Substances by Capillary Electrophoresis ChaeXuan Zhang, Frans von Heeren, and Wotfgang IhonnBnn* Department of Clinical Phamacology, University of Bem, Murtenstrasse 35, CH-3010 Bem, Swikerland

"he electrokinetic separation behavior of positively chargeable substances with various hydrophobicities over the pH range 2-11 is described, using plain aqueous buffers, binary media with organic solvents, and systems with surfactantsat concentrationsbetween 0.0005and 75 mM or other buffer additives. Examples studied include dextromethorphan, methadone, ketoconazole, itraconazole, amiodarone, and desethylamiodarone, compounds that play an important role in pharmacotherapy. Electrokinetic chromatography systems comprising dynamically formed micelles or ionic buffer additives that can form ion pairs with the solutes are shown to be unsuitable for the separationof highly hydrophobic, positively c h g e able compounds, including the latter three drugs investigated. However, capillary zone electrophoresis (CZE) in binary systems with organic solvents (40-80%, v/v) at a buffer pH near or lower the solute's p& values or CZE at low pH in the presence of an electrically neutral complexing agent, such as /I-cyclodextrh, can be employed effectively. Data presented illustrate that P g a values in binary systems with organic solvents that are more basic than water are smaller than those in aqueous media. Furthermore, somewhat less hydrophobic and positively chargeable compounds can be su5iciently dissolved in aqueous buffer at pH < P g a and can therefore be analyzed by CZE without any buffer additives. Capillary electrophoresis (CE) has been shown to be a powerful analytical tool for a wide range of samples, including many applications to the analysis of drugs.1s2 The electrokinetic separation behavior of hydrophobic drugs is not yet fully understood. Using micellar electrokinetic chromatography (MEKC), many hydrophobic compounds have been shown to be totally incorporated into the micelles and thus cannot be separated. To reduce the large values of the capacity factors, ~ r e a ~and -~ ~yclodextrin~~~ have been added to the micellar solutions, as well as bile salts6of weaker solvation capability compared to dodecyl sulfate, have been employed. The addition of organic solvents (1) Guzman, N. A, Ed. Capillay Electrophoresis Technology; Marcel Dekker: New York, 1993. (2) Thormann, W.; Molteni, S.; Caslavska, J.; Schmutz, A Electrophoresis 1994, 15, 3-12. (3) Terabe, S.; Ishihama, Y.; Nishi, H.; Fukuyama, T.; Otsuka, IC]. Chromafogr. 1991,545, 359-168. (4) Terabe, S.; Miyashita, Y.; Ishihama, Y.; Shibata, 0.]. Chromafogr. 1993, 636,47-55. (5) Terabe, S.;Miyashita, Y.; Shibata, 0.; Barnhart, E. R; Alexander, L. R; Patterson, D. G.;Karger, B. L;Hosoya, IC;Tanaka, N.]. Chromatogr. 1990, 516,23-31. (6) Nishi, H.; Fukuyama, T.; Matsuo, M.; Terabe, S.]. Chromafogr. 1990,513, 279-295.

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has also been shown to reduce the capacity factor of a and the separation of hydrophobic polyaromatic hydrocarbons by MEKC with 30-50% (v/v) organic solvents has been described.1° With ionic buffer additives, such as tetraalkylammoniumi o n ~ ~ l - ~ ~ or surfactantd4 to buffers containing typically 50% (v/v) of an organic solvent, single molecules are acting as carriers and separation is based upon difEerences in the interaction of neutral solutes with the ionic carriers. This technique can be called ionic electrokinetic chromatography, another mode of electrokinetic ~hromatography.~~ With these techniques, nice separations for selected classes of molecules have been achieved. As of today, however, no general rules for the selection of CE separation conditions have been elucidated. Thus, failures in attempted separations are mostly based upon inadequate use of a running buffer.16 Thus far, CE investigations dealing with hydrophobic s u b stances have mostly been focused on electrically neutral solutes. The work described here is concerned with the electrokinetic separation behavior of positively chargeable, highly hydrophobic drugs, including itraconazole, amiodarone, and desethylamiodarone Figure 1). Moderately hydrophobic drugs (ketoconazole, methadone, dextromethorphan), as well as three hydrophilic compounds (theophylline, caffeine, naproxen), have also been investigated. These compounds represent pharmaceuticals that are widely applied for pharmacotherapy, and with the exception of naproxen, they have nitrogen in their chemical structures (Figure 1) and are therefore positively chargeable. Although CE methods for the determinations of methadone,17dextromethorphan,I8the~phylline,'~*~~ and naproxen20,21 were previously developed for clinical purposes, these compounds have been included to provide better insight into selection of separation conditions. (7) Balchunas, A T.; Sepaniak, M. J. Anal. Chem. 1988,60, 617-621. (8) Thormann, W.; Lienhard, S.; Wemly, P. J. Chromafogr. 1993,636,137148. (9) Kaneta, T.;Yamashita, T.; Imasaka, T. Electrophoresis 1994,15, 12761279. (10) Otsuka, IC; Higashimori, M.; b i k e , R.; Karuhaka, IC; Okada, Y.; Terabe, S. Electrophoresis 1994,15,1280-1283. (11) Walbroehl, Y.; Jorgenson, J. W. ]. Chromafogr. 1984,315,135-143. (12) Walbroehl, Y.; Jorgenson, J. W. Anal. Chem. 1986,58, 479-481. (13) Shi, Y.; Fritz, J. S. ]. High Resoluf. Chromatogr. 1994,17, 1-6. (14) Ahuja, E. S.; Foley, J. P. ]. Chromafogr. 1994,680, 73-83. (15) Terabe, S. In Capillny ElecfrophoresisTechnologv;Gurman, N. A, Ed.; Marcel Dekker: New York, 1993; pp 66-88. (16) Bmmley, W. C.; Jones, W. J. J. Chromatogr. 1994,680, 163-173. (17) Molteni, S.; Caslavska, J.; Allemann, D.; Thormann, W. J. Chromatogr. 1994, 658, 355-367. (18) Caslavska, J.; Hufschmid, E.; Theurillat, R; Desiderio, C.; Wolfisberg, H.; Thormann, W.]. Chromatogr. 1994,656,219-231. (19) Thormann, W.; Minger, A; Molteni, S.; Caslavska, J.; Gebauer, P. /. Chromufogr. 1992,593,275-288. (20) Zhang, C.-X.; Thormann, W. J. Capillaty Electrophoresis 1994,1, 208-218. (21) Schmutz, A; Thormann, W. Elecfrophoresis 1994,15,1295-1303. 0003-2700/95/0367-2070$9.00/0 0 1995 American Chemical Society

Theophylline

Caffeine

Naproxen

Dextromethorphan

Methadone

Ketoconazole

Amiodarone

Derethylamiodarone

"'~(clucl P

L.0

0

~ c ~ . - . 8 N ~U N 0 N ~ ~ - E r " l - " ~

ltraconazole Flgure 1. Chemical structuresof the drugs investigated in this work.

EXPERIMENTAL SECTION

Chemicals and Drugs. All chemicals were of analytical reagent grade and the organic solvents methanol, ethanol, l-propanol, 2-propanol, acetonitrile, and tetrahydrofuran (THF)were of HPLC grade. Sodium dodecyl sulfate (SDS) was purchased from Bioprobe System (Montreuil-Sous-Bois,France), j?-cyclodextrin V-CD) from Fluka (Buchs, Switzerland), sodium taurocholate (STC) from Calbiochem (La Jolla, CN, sodium deoxycholate from Merck (Darmstadt, Germany), cetyltrimethylammonium bromide (CTAB) from S i a (St. Louis, MO), and urea from Aristar (BDH Limited, Poole, Great Britain). Itraconazole and ketoconazole were obtained from Janssen Research Foundation (Beerse, Belgium), and naproxen from Griinenthal (Mitlodi, Switzerland). Theophylline, caffeine, and methadone hydrochloride were of European Pharmacopoeia quality and were supplied by the university hospital pharmacy (Bern, Switzerland). Dextromethorphan hydrobromide was a kind gift of F. HoffmannLa Roche (Basel, Switzerland). Amiodarone hydrochloride and desethylamiodarone hydrochloride were received from Sanofi (Basel, Switzerland). Preparation of Sample Solutions and Running ButTers. Stock solutions of drugs were prepared in different solvents, namely, in THF for itraconazole (0.49 mg/mL); in methanol for amiodarone hydrochloride (0.61 mg/mL), desethylamiodarone hydrochloride (0.79 mg/mL), and naproxen (1.37 mg/mL); in 0.04 M phosphate buffer (PH 2.2) for ketoconazole (0.96 mg/mL); or in water for methadone hydrochloride (1.22 mg/mL), dextromethorphan hydrobromide (1.86 mg/mL), theophylline (0.76 mg/mL), caffeine (0.75 mg/mL), and phenol (4.68 mg/mL). The sample solutions were prepared by adding appropriate aliquots of the stock solutions to methanol, resulting in solute concentrations of 30-130 pg/mL. The phosphate running buffer was prepared by adding appropriate aliquots of 0.2 M H3P04, 0.2 M NaHzP04, 0.2 M Na2HP04, and 0.1 M NaOH stock solutions into water or into mixtures of water and organic solvents. Additives such as surfactants, cyclodextrin, or urea were directly dissolved

in the running phosphate buffers. The pH of buffers without organic solvents was determined by a Ross combination pH electrode (Orion, Boston, MA). For the binary systems, the pH value given refers to that of an aqueous buffer with the same phosphate composition. Capillary Electrophoresis. CE was carried out in two instruments. The experiments with organic solvents were performed on a BioFocus 3000 capillary electrophoresis system @ioRad Labs, Hercules, CN and all other experiments on a Prince apparatus (Lauerlabs, Emmen, The Netherlands). In both instruments, a constant voltage of 20 (anode on sampling end) or -20 kV (cathode on sampling side) was applied, and absorbance detection toward the capillary end was effected at 220 nm. In the Prince instrument, a fused-silica capillary (Polymicro Technologies, Phoenix, AZ) of 50 pm i.d. and 44.5 cm (55.5 cm) effective (total) length was employed. The temperature of the capillary was kept at 35 "C. Capillary conditioning was effected by rinsing with running buffer for 5 min using a positive pressure of 3000 mbar. Sample injection was carried out by applying a positive pressure of 20 mbar for 0.1 min. The signal from the UV absorbance detector was recorded by a Model D-2000 chromatointegrator (Merck-Hitachi, Darmstadt, Germany). For the experiments with the BioFocus 3000, the capillary (Polymicro Technologies) was of 50 pm i.d. and 35.4 cm (40.0 cm) effective (total) length. The temperatures of capillary and sample carousel were kept at 20 "C. Sample injection occurred by a positive pressure at 5 psi (4 psi-s). Capillary conditioning between runs was effected by rinsing with 0.1 M NaOH (1 min) and running buffer (2 min) applying positive pressure of -100 psi. The electropherograms were evaluated using the BioFocus 3000 postrun software version 3.10 @io-Rad). RESULTS AND DISCUSSION

Capillary Zone Electrophoresisin Aqueous Solution. All drugs investigatedhave one or several nitrogen-containing groups or a carboxylic function (naproxen) in their chemical structures F i r e 1). Thus, depending on pH, these compounds can be charged, and thereby possibly separated on the basis of differences in electrophoreticmobilities. The electrophoreticbehavior of all nine drugs was studied in the pH range 2-9, the pH dependence of the determined electrophoretic mobilities being shown in Figure 2. The effective electrophoretic mobility (for definition refer to ref 22) was calculated using the solvent peak as a marker of the electroosmotic A positive (negative) electrophoretic mobility means that the solute has a positive (negative) charge and migrates electrophoretically toward the cathode (anode). The electrophoreticbehavior observed is closely related to the durgs' structures (Figure 1). At pH >6.8 theophylline (8) was found to have a negative charge, which can be ascribed to the dissociation of hydrogen in the imidazole ring. Caffeine (7) does not have such a proton and therefore no negative mobility was observed for this substance. The sharp change in theophylline mobility between pH 7 and 9 is in agreement with the reported (22) Karger, B. L.; Foret, F. In Capillay Electrophoresis Technology;Guman, N. A, Ed.;Marcel Dekker: New York, 1993; pp 3-64. (23) Quang, C.; Strasters, J. K; Khaledi, M. G. Anal. Chem. 1994, 66, 16461653. (24) Copper, C. L.; Sepaniak, M. J. Anal. Chem. 1994, 66, 147-154.

Analytical Chemistry, Vol. 67, No. 13, July 1, 1995

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40

1

1

4* 5

IS

P* I

I

N

E O

0

5

10

15

20

Time (min) Figure 3. Typical electropherograms obtained with the Prince instrument. The running buffers were (A) 0.04 M phosphate solution (pH 2.2) and (B) the same as in (A) and containing 20 mM p-CD. The applied voltage was 20 kV, and the current was -30yA. Other conditions as in the Experimental Section. The symbol “s” indicates the solvent peak. The key is given in Figure 2 .

-20

-30 0

2

6

4

0

10

PH Figure 2. pH dependence of the effective electrophoretic mobility in 0.04 M aqueous phosphate buffer. The data were obtained with the Prince instrument. The applied voltage was a constant 20 kV, and depending on pH, the currents were between 26 and 72pA. Other conditions are given in the Experimental Section. Key: 1, ketoconazole; 2, dextromethorphan; 3, methadone; 4, desethylamiodarone; 5, amiodarone; 6, itraconazole; 7 , caffeine; 8, theophylline; 9, phenol; 10, naproxen.

pKa value of MZ5 and with the pHdependent elution in MEKC with SDSZ6The pKa for the cationic acid is 2.5,25explaining the fact that small positive mobilities were observed for both theophylline and caffeine at pH