Anal. Chem. 1996, 68, 1028-1032
A Hydrophobicity Scale Based on the Migration Index from Microemulsion Electrokinetic Chromatography of Anionic Solutes Yasushi Ishihama,* Yoshiya Oda, and Naoki Asakawa
Department of Analytical Chemistry and Pharmaceutical Research, Tsukuba Research Laboratories, Eisai Co., Ltd., 5-1-3 Tokodai, Tsukuba, Ibaraki 300-26, Japan
A migration index (MI) concept, previously introduced as a novel scale for measuring the hydrophobicity of neutral solutes, was applied to anionic solutes. The required parameters for determining the MI were measured by both microemulsion electrokinetic chromatography with a sodium dodecyl sulfate/1-butanol/heptane/buffer solution and capillary zone electrophoresis with 1-butanol added to the buffer. The obtained MI values of anionic solutes correlated very well with the logarithm of the partition coefficients between 1-octanol and water (log P), whereas the reversed-phase chromatographic retention parameter (log k′w), which is also used as a hydrophobicity scale, correlated very little with log P for the tested anionic solutes. Moreover, the linear relationship between the MI and log P of the anionic solutes was consistent with that of neutral solutes. Although the relationship between the MI and log P of solutes having carboxylic groups deviated from those of others, the MI provided better regression to bioactivities such as human skin permeability than log P in quantitative structure-activity relationship studies. Therefore, this system would be an appropriate biochemical model. In addition, because measurement of the MI is easy, rapid, and reproducible, and the required sample amount is nanogram size, the MI is expected to be a useful and universal hydrophobicity scale. It is well known that the hydrophobicity of drugs affects their absorbability and transportation in the body because the wall of the alimentary tract and the target tissues of the drugs consist of highly hydrophobic components such as phospholipids and proteins.1 Thus, conventional hydrophobicity scales, such as the logarithm of the partition coefficients between 1-octanol and water (log P) and the logarithm of the capacity factors in reversed-phase HPLC (log k′w) are widely used for developing new drugs with quantitative structure-activity relationship (QSAR) studies.2,3 However, the former has some disadvantages, such as the amount of time consumed in taking the measurement and the low reproducibility of the measurements,4 and the latter requires some corrections for hydrogen-bonding effects.5 Moreover, it is questionable to use both of these two-phase systems as biochemical models. Liposomes,6,7 cells,6 and micelles8 have amphiphilic and anisotropic properties in analogy to biomembranes. However, it (1) Goth, A. Medical Pharmacology; Mosby: St. Louis, MO, 1981. (2) Leo, A.; Hansch, C.; Elkins, D. Chem. Rev. 1971, 71, 525. (3) Kalizan, R. Anal. Chem. 1992, 64, 619A. (4) Cichna, M.; Markl, P.; Huber, J. F. K. J. Pharm. Biol. Anal. 1995, 13, 339. (5) Yamagami, C.; Yokota, M.; Takao, N. J. Chromatogr. A 1994, 662, 49.
1028 Analytical Chemistry, Vol. 68, No. 6, March 15, 1996
has been difficult and tedious to monitor the partitioning of solutes in these models. Capillary electrophoresis (CE) is a powerful tool for determining some of the physicochemical properties, such as the acid dissociation constants9 and protein-ligand binding constants.10,11 Recently, the partitioning behavior of solutes in micelles was evaluated by electrokinetic chromatography (EKC), which is a branch of CE.12-14 However, electrostatic interaction and hydrogenbonding effects between solutes and ionic micelles were observed. On the other hand, microemulsion electrokinetic chromatography (MEEKC) with a sodium dodecyl sulfate (SDS), 1-butanol, heptane, and water solution provided a novel hydrophobicity scale, a migration index (MI), for neutral solutes.15 The MI has a linear relationship to log P and this system correlated more thermodynamically with the liposome system, which is a more reasonable biochemical model than the octanol-water system. Preliminary results of QSAR studies demonstrated that a MI was a better biological hydrophobicity scale than log P. However, this method could not be applied to ionic solutes because of difficulties encountered in measuring the electrophoretic mobilities of the solute in the aqueous phase of MEEKC (µaq). In this work, a MI was applied to anionic solutes. The mobility was experimentally determined, and the appropriation of the obtained values was evaluated. For solutes with a negative charge at pH 7.0, the MI was determined and compared to conventional hydrophobicity scales such as log P and log k′w. In addition, to clarify the biological meaning of a MI, QSAR studies were performed on some bioactivities. EXPERIMENTAL SECTION MEEKC was performed using a P/ACE 2100 system (Beckman Instruments, Fullerton, CA). The preparation of the microemulsion was previously reported.15 All samples were purchased (6) Davis, S. S.; James, M. J.; Anderson, N. H. Faraday Discuss. Chem. Soc. 1986, 81, 313. (7) Katz, Y.; Diamond, J. M. J. Membr. Biol. 1974, 17, 101. (8) (a) Treiner, C. J. Colloid Interface Sci. 1983, 93, 33. (b) Treiner, C.; Chattopadhyay, A. K. J. Colloid Interface Sci. 1986, 109, 101. (9) Ishihama, Y.; Oda, Y.; Asakawa, N. J. Pharm. Sci. 1994, 83, 1500. (10) Honda, S.; Taga, A.; Suzuki, K.; Suzuki, S.; Kakehi, K. J. Chromatogr. 1992, 597, 377. (11) Kraak, J. C.; Busch, S.; Poppe, H. J. Chromatogr. 1992, 608, 257. (12) Ishihama, Y.; Oda, Y.; Uchikawa, K.; Asakawa, N. Chem. Pharm. Bull. 1994, 42, 1525. (13) Chen, N.; Zhang, Y.; Terabe, S.; Nakagawa, T. J. Chromatogr. A 1994, 678, 327. (14) Herbert, B. J.; Dorsey, J. G. Anal. Chem. 1995, 67, 744. (15) Ishihama, Y.; Oda, Y.; Uchikawa, K.; Asakawa, N. Anal. Chem. 1995, 67, 1588. 0003-2700/96/0368-1028$12.00/0
© 1996 American Chemical Society
Figure 1. Dependence of (A) k′ and (B) MI on the SDS concentration in microemulsion solutions. Samples: 1, pyrazine; 2, methylpyrazine; 3, resorcinol; 4, benzyl alcohol; 5, phenol; 6, benzaldehyde; 7, nitrobenzene; 8, benzene; 9, p-chlorophenol; 10, toluene; 11, p-propylphenol; 12, ethylbenzene; 13, propylbenzene. Separation solutions: SDS/6.49% 1-butanol/0.82% heptane (wt %) in 0.1 M borate-0.05 M phosphate (pH 7.0); capillary, 50 µm i.d. × 27cm; voltage, 7.5 kV; detection, 214 nm; temperature, 25 °C.
from Sigma (St. Louis, MO), Wako (Osaka, Japan), and Tokyo Kasei Kogyo (Tokyo, Japan). All other reagents were of analytical grade. MS-Excel v. 4.0 (Microsoft, Redmond, WA) with a Macintosh Quadra 650 personal computer (Apple Computer, Cupertino, CA) was used to run a multiline fitting program. The usual “shake flask” method was carried out for the measurement of log P between 1-octanol and a borate-phosphate buffer (pH 7.0). On the other hand, log P values of neutral forms of solutes were obtained from the list of measured values in the database of Mac-ClogP v. 1.0.3 (BioByte Corp., Claremont, CA).16 The log k′w from the reversed-phase HPLC method was measured according to the reported procedure,5 in which the employed column was a CAPCELL PAK C18 (4.6 mm × 150 mm; Shiseido, Tokyo, Japan), and the mobile phases consisted of methanol and the buffer that was used in the microemulsion solution. The log k′w was obtained by extrapolating the log k′ to 0% methanol mobile phase. The determination of the MI is described in the next section.
in the aqueous phase and microemulsion phase, respectively, and µeff is the effective mobility in the microemulsion solution. The migration index, MI, is given by
MI ) a log k′ + b where a and b are the slope and the intercept of a calibration line between the MI values of the reference solutes and their log k′, respectively. In this work, the series of reference solutes consisted of benzaldehyde (MI ) 4.5), benzene (6.0), toluene (7.0), ethylbenzene (8.0), propylbenzene (9.0), and butylbenzene (10.0). Thus, MI is given by
MI ) a log
k′ )
µaq - µeff µeff - µme
(1)
where µaq and µme are the electrophoretic mobilities of the solute (16) Leo, A. J. Chem. Rev. 1993, 93, 1281.
)
µaq - µeff +b µeff - µme
(3)
or µeff is given by
µeff ) RESULTS AND DISCUSSION Determination of µaq. The capacity factor, k′, of an ionic solute in MEEKC is defined as
(
µaq + µme{10(MI - b)/a} 1 + 10(MI - b)/a
(4)
Among the variables in eq 3, µeff, µme, a, and b can be obtained by measuring the migration times of the analyte, methanol, dodecylbenzene, and the reference standards. However, µaq cannot be measured with a single MEEKC analysis. Therefore, to obtain µaq, it was assumed that the MI and µaq are constant even if the volume of the microemulsion phase changes. When these assumptions are valid, the MI can be calculated by changing the volume of the microemulsion phase. In micellar EKC with SDS, Muijelaar et al. reported that the retention indices were constant Analytical Chemistry, Vol. 68, No. 6, March 15, 1996
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Table 1. Dependence of log P on the Concentration of Some Carboxylic Acids sample concn (µg/mL) sample
4
20
200
salicylic acid proglumide naproxen flurbiprofen ibuprofen indomethacin
-1.30 -0.23 0.57 1.52 1.72 1.49
-1.48 -0.25 0.57 1.25 1.53 1.44
-1.62 -0.32 0.52 1.19 1.28 1.26
Table 2. Skin Permeability and Other QSAR Parameters of Non-Steroidal Antiinflammatory Drugs
Figure 2. Electrophoretic mobilities of anionic solutes in aqueous phases measured by different methods. Samples: indomethacin, salicylic acids, proglumide, warfarin, and naproxen. Conditions: capillary, 50 µm i.d. × 27 cm; voltage, 7.5 kV; detection, 214 nm; temperature, 25 °C. Separation solutions: (a) 0.1 M borate-0.05 M phosphate (pH 7.0), (b) 0.1 M borate-0.05 M phosphate (pH 7.0) including 8.0% (v/v) 1-butanol, (c) 0.1 M borate-0.05 M phosphate (pH 7.0) including 8.0% (v/v) 1-butanol and 50 mM NaCl.
sample
log Ra
MI
log P
log P ′ (pH 7.0)b
aspirin bufexamac diclofenac flufenamic acid flurbiprofen ibuprofen indomethacin ketoprofen salicylic acid salicylamide
-0.69 -0.65 -1.00 -1.50 -0.50 -0.52 -1.00 -0.10 0.38 -0.37
5.15 6.58 6.42 7.81 5.64 5.80 6.43 5.20 2.39 4.45
1.19 2.45 4.40 5.25 4.16 3.50 4.27 3.12 2.26 1.28
-1.70 2.21 1.58 2.41 1.25 1.53 1.44 0.33 -1.48 1.28
a According to Yano et al.21 R ) % abs/(100 - %abs). b The sample concentration was 20 µg/mL.
Figure 3. Relationship between log P and the MI for neutral and anionic solutes. Samples: O, sulfamerazine, sulfamethazine, sulfaquinoxaline, warfarin, p-hydroxybenzaldehyde, p-nitrophenol, methylparaben, ethylparaben, and propylparaben; b, 53 neutral solutes in ref 15.
when the volume of the micellar phase changed.17 In this study, the effect of the SDS concentration on the partition of the neutral solutes, including hydrogen bond acceptors and donors, was studied (Figure 1). The MI was constant, although the k′ increased as the SDS concentration increased. Thus, in order to measure the MI of anionic test solutes, three MEEKC separation solutions with different SDS concentrations were prepared. The MEEKC separations were performed with each separation solution, and a multiline fitting program was run to obtain the MI and µaq using eq 4. On the other hand, the effective mobilities of these solutes were measured by capillary zone electrophoresis (CZE) with three different buffers and were compared with the (17) Muijselaar, P. G. H. M.; Claessens, H. A.; Cramers, C. A. Anal. Chem. 1994, 66, 635.
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calculated µaq. As shown in Figure 2, the effective mobilities in the 0.1 M borate-0.05 M phosphate buffer (pH 7.0) that included 8% (v/v) 1-butanol solution were in fair agreement with the calculated µaq. Because the tediousness of the calculation method was not suitable for the development of a rapid and easy measurement of the hydrophobicity, the effective mobilities measured by this CZE method were employed in eq 3 as µaq instead of the calculated µaq for the determination of the MI. In addition, slight errors might be inherent in the calculation of the MI and µaq because the MI of every solute might not always be independent of the SDS concentration. Determination of the MI and Correlation with log P or log k′w. For neutral solutes, the MI correlated with log P without considering hydrogen-bonding effects, as previously reported.15 The MI for nine test compounds with a negative charge at pH 7.0 were measured by the following procedure: µaq was measured by CZE with the borate-phosphate buffer (pH 7.0) including 8% (v/v) 1-butanol, and µeff, µme, a, and b were calculated from the migration times of the analyte, methanol, dodecylbenzene, and the reference standards, which were measured by MEEKC with 1.66% (w/w) SDS, 6.49% (w/w) 1-butanol, 0.89% (w/w) heptane, and the borate-phosphate buffer (pH 7.0). The MI values were determined by substituting the values obtained for µaq, µeff, µme, a, and b into eq 3. The log P values between 1-octanol and the borate-phosphate buffer (pH 7.0) were measured to compare with MI (Figure 3). For these anionic solutes, the MI also correlated to log P. Furthermore, the linear relationship was almost the same as that for neutral compounds, and no electrostatic repulsion was observed between the anionic solutes and SDS. This might be because the surface charge of the microemulsion was sufficiently shielded by 1-butanol, as previously reported.15 Another hydrophobicity scale commonly used is log k′w which is determined by reversed-phase HPLC. This scale has better reproducibility than
Figure 4. Correlation of log P with (A) log k′w and (B) MI. Samples: sulfamerazine, sulfamethazine, sulfaquinoxaline, warfarin, p-hydroxybenzaldehyde, p-nitrophenol, methylparaben, ethylparaben, and propylparaben. Table 3. QSAR Parameters for Substituted Phenols sample
MI
log P
resorcinol p-methoxyphenol phenol p-fluorophenol p-ethoxyphenol p-nitrophenol p-cresol o-cresol m-cresol methylparaben p-chlorophenol p-ethylphenol p-bromophenol 2-naphthol p-tert-butylphenol
3.12 4.02 4.16 4.81 4.81 3.89 5.17 5.09 5.09 4.88 6.26 6.36 6.51 6.77 7.86
0.78 1.34 1.46 1.77 1.81 1.91 1.94 1.95 1.96 1.96 2.39 2.58 2.59 2.84 3.31
log Kpa
-log AH50b
equation
r
s
F
log Kp ) -0.205 MI2 + 2.54 MI - 12.9 log Kp ) -0.514(log P)2 + 2.87 log P - 9.04 -log AH50 ) 0.449MI + 0.426 -log AH50 ) 0.914 log P + 0.958
0.965
0.183
54.7
2.222 2.108 2.523 2.796
0.958
0.200
45.0
0.977 0.976
0.130 0.134
-7.18 -5.64
-5.81 -5.31 -5.36 -5.37 -5.60 -5.00 -5.01 -5.00 -5.11
Table 4. Regression Analyses
149 140
2.854 2.620 3.131 3.444 3.896
a K is human skin permeability constant in cm s-1 from ref 22. bAH p 50 is the concentration which inhibited osmotic hemolysis by 50% from ref 23.
log P by the shake flask method, requires only a nanoscale sample amount, and can be automatically determined. In Figure 4, the relationships between these three scales are shown. For the anionic solutes employed in this study, log k′w correlated less with log P, because this HPLC system is different from the 1-octanolwater system in terms of recognizing electron-donating and -accepting solutes. On the other hand, the MI has the same advantages as log k′w in measuring and hydrogen-bonding effects do not need to be considered. Intermolecular Effect of Solutes. Some carboxylic acids did not satisfy the relationship between the MI and log P of neutral solutes. Moreover, the dependency of their log P values on their solute concentrations was observed (Table 1). As their concentrations increased, their log P values decreased. This might be (18) Smith, R.; Tanford, C. Proc. Natl. Acad. Sci. U.S.A. 1973, 70, 289.
caused by the association of solute molecules. In fact, it is well known that carboxylic acids associate to form aggregates of two molecules by hydrogen bonding, which results in a change in the distribution between the two phases.18 On the other hand, the MI values of the carboxylic acids were independent of the concentration of the solutes. In the microemulsion solution, SDS monomers might prevent solutes from associating with each other. It might be important to the drug bioactivity that their hydrophobicity changes with the association of solutes.19,20 However, it was difficult to measure log P values of solutes forming aggregates by the shake flask method, while the MI has a unique value independent of the solute concentration. Quantitative Migration-Activity Relationship (QMAR). To investigate the biological meaning of the MI values in comparison with log P, a QMAR study on human skin permeability was performed for non-steroidal antiinflammatory drugs (NSAIDS), whose MI and log P values deviated from the linear relationships between the MI and log P of other compounds that have no carboxylic group, as mentioned above. The QMAR approach involves performing the QSAR using migration data measured by capillary electrophoresis instead of a conventional hydrophobicity scale, as previously introduced.15 The QMAR results are listed in Table 2. The MI was compared with log P at pH 7.0 (log P ′) (19) Finkelstein, A. Biochim. Biophys. Acta 1970, 205, 1. (20) Celis, H.; Estrada-O, S.; Montal, M. J. Membr. Biol. 1974, 18, 187.
Analytical Chemistry, Vol. 68, No. 6, March 15, 1996
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Figure 5. Relationships between human skin permeability and three hydrophobicity parameters, such as (A) MI, (B) log P ′, and (C) log P, for non-steroidal antiinflammatory drugs.
at a 20 µg/mL solute concentration and log P of the neutral form, which were from the list of measured values in the ClogP database,16 whereas estimated values were employed in the original paper.21 As shown in Figure 5, the MI correlated very well with the bioactivity, while log P ′ and log P correlated much less. The results of the regression analyses were as follows:
log R ) -0.0230MI2 - 0.100MI + 0.694 r2 ) 0.854 s ) 0.222 f ) 41.3 log R ) -0.186(log P)2 + 0.924 log P - 1.335 r2 ) 0.687 s ) 0.329 F ) 7.68 log R ) 0.141(log P ′)2 - 0.160 log P ′ - 0.085 r2 ) 0.555 s ) 0.392 F ) 4.37 where r is the correlation coefficient, s is the standard deviation from regression, and F is the value of the F-ratio between regression and residual variances. The MI scale was a biologically (21) Yano, T.; Nakagawa, A.; Tsuji, M.; Noda, K. Life Sci. 1986, 39, 1043. (22) Robert, M. S.; Anderson, R. A.; Swarbrick, J. J. Pharm. Pharmacol. 1977, 29, 677. (23) Machleidt, H.; Roth, S.; Seeman, P. Biochim. Biophys. Acta 1972, 225, 178.
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appropriate hydrophobicity scale, differing from the conventional log P scale. Other QSAR studies were performed for substituted phenols (Table 3). In these cases, the MI also provided slightly better results than conventional log P (Table 4). In conclusion, the MI values of anionic solutes were easy to determine compared with those of neutral solutes. Moreover, the obtained hydrophobicity scale was the same as that for neutral solutes in terms of the correlation with log P. For carboxylic acids although the relationship between the MI and log P deviated from that of other compounds, the MI values correlated more quantitatively with the bioactivity than log P. Therefore, the MI would be a more useful scale for expressing solute hydrophobicity biologically. Although samples and the microemulsion were limited to anionic species in this work, further studies on cationic or amphoteric solutes and other microemulsions are in progress in our laboratories.
Received for review December 21, 1995.X
October
17,
1995.
Accepted
AC9510402 X
Abstract published in Advance ACS Abstracts, February 1, 1996.
