A full study on the application of MMEEKC was recently published: Wong K-S, Kenseth J, Strasburg R: Validation and Long-Term Assessment of an Approach for the High Throughput Determination of Lipophilicity (log Pow) Values Using Multiplexed, Absorbance-Based Capillary Electrophoresis. J Pharm Sci 2004, 93:916-931.

-Application-

High Throughput Estimation of Octanol-Water Partition Coefficients (log Pow Values) by Multiplexed-Microemulsion Electrokinetic Chromatography with the MCE 2000™ System Kit-Sum Wong, Ph.D. Jeremy Kenseth, Ph.D. Introduction The partition coefficient (P) and the dissociation constant (Ka) are physicochemical properties often determined during early drug discovery and development. P is defined as the equilibrium concentration ratio of a solute between a lipid phase and an aqueous phase:

P=

[ S ]oil [ S ]water

(1)

In particular, the octanol–water partition coefficient (log Pow value) is commonly used as a means for defining the relative lipophilicity of a drug. Log Pow values can be utilized to predict drug transport properties across cell membranes and have been applied to quantitative structureactivity relationships (QSARs).1 Recently, microemulsion electrokinetic chromatography (MEEKC) has been successfully applied for the indirect evaluation of A microemulsion is an log Pow values.2-6 optically transparent solution composed of oil droplets surrounded by surfactant and cosurfactant.7 In MEEKC, the migration time of a solute depends on its partitioning behavior between the microemulsion and aqueous phases of the solution. For a neutral solute, the capacity factor k’ can be calculated according to following equation:

k' =

t S − t EOF t EOF (1 − t S /t ME )

(2)

where tEOF, tS, and tME are the migration times of the electroosmotic flow, solute and

microemulsion, respectively. The relationship between the partition coefficient and the capacity factor can be expressed as the following: log Pow = a log k’ + b

(3)

where a and b are the slope and the intercept of a calibration curve constructed from experimentally determined log k’ values of reference solutes and their known literature log Pow values. The MCE 2000™ system is a multiplexed, absorbance-based, capillary electrophoresis instrument that separates and analyzes compounds. It is capable of the simultaneous analysis of up to 96 samples and has been successfully applied to high throughput analysis of compound log Pow values.8-10 This application note describes the use of the MCE 2000™ system for estimating log Pow values using multiplexed-MEEKC (MMEEKC). Experimental The MMEEKC experiments were carried out on a MCE 2000™ instrument (CombiSep, Ames, IA, USA) with UV detection at 214 nm. A 96capillary array composed of fused-silica capillaries (75 µm i.d., 50 cm effective length, 76 cm total length) was used for the study. The new capillary array was conditioned with 0.1 N NaOH for 5 min, water for 10 min and microemulsion buffer for 10 min in sequence. The capillary array was rinsed with microemulsion buffer for 10 min between analyses, washed 3 times with deionized water for 5 min each at the end of the day, and rinsed with 0.1 N HCl as needed to restore separation

efficiency. The microemulsion was prepared by mixing 6.61% (w/v) 1-butanol, 0.81% (w/v) nheptane, 3.31% (w/v) sodium dodecylsulfate, and 800 mL 68 mM CAPS buffer at pH 10.3 via sonification for 30 min. The mixture was allowed to stand at room temperature for 1 h, diluted to volume with CAPS buffer and filtered through a 0.45-µm nylon membrane filter. Standard mixture compounds and solutes (0.5 mg/mL or 0.5 µL/mL) were dissolved in the microemulsion along with DMSO (2.5 µL/mL) and dodecylbenzene (0.5 µL/mL) via sonification. Samples were arranged in a 96-well plate and injected by applying a vacuum at -0.2 psi for 20s to 25s. Separations were performed at 6.5 kV with a VACE (vacuum-assisted CE) level of –0.1 psi. MCE Manager™ software (CombiSep, Inc.,

Ames, IA) was employed for MCE 2000™ system control and data collection/processing. Results and Discussion MMEEKC Figure 1 shows representative 96-capillary MMEEKC electropherograms obtained for a standard mixture and 23 different sample solutions (analyzed in quadruplicate) using the MCE 2000™. Typically, the standard mixture and 23 samples (DMSO, solute, dodecylbenzene) were loaded in 4 consecutive wells of a 96-well plate. The first and last peaks of each electropherogram were the EOF marker and microemulsion marker, respectively.

Figure 1. MCE 2000™ log Pow analysis: 96-capillary MMEEKC electropherograms.

Calculation of MMEEKC log k’ and log Pow values The standard mixture was composed of 6 solutes (Table 1) that covered a wide range of literature log Pow values from -0.3 to 4.8.11 The mixture was used as a calibration standard and analyzed with the sample solutes in every run. Compound pyrazine benzamide nicotine quinoline 4-Propylphenol imipramine

Literature log Pow -0.26 0.64 1.17 2.03 3.20 4.42

The MCE log P Calculator™ program was used to process electropherograms generated by the MCE Manager™. There are two tab-controlled screens in the MCE log P Calculator™ program. In the Calibration screen (Figure 3), the program constructs a calibration curve (log P = A × log k’ + B) based on the average MMEEKC log k’ values of each standard compound from the four standard mixture electropherograms and their entered literature log Pow values. A calibration equation of log Pow = 1.7302 log k’+ 1.1572 (R2 = 0.9873) was obtained for the run analyzed in Figure 3.

Table 1. Literature log Pow values of solutes in the standard mixture.

Figure 3. Data analysis using the MCE log P Calculator™ program: Calibration screen.

The A and B values are then utilized in the log P screen to calculate the MMEEKC log Pow values of the other solutes, based on their MMEEKC log k’ values as determined in the same run (Figure 4). The calculated

MMEEKC log k’ values and MMEEKC log Pow values of solutes using the MCE log P Calculator™ program are summarized in Table 2.

Figure 4. Data analysis using the MCE log P Calculator™ program: MMEEKC log Pow determination.

a

Solute

acebutotol HCl 1-aminonaphthalene 2-aminopyridine aniline benzamide caffeine 4-chloroaniline chlorthalidone coumarin m-cresol ethyl p-aminobenzoate ethylbenzoate hydroquinine 4’-hydroxyacetophenone indazole lidocaine naphthalene nefopam HCl nicotine nitrobenzene 4-nitrophenol phenyl acetate 4-propylphenol pyrazine pyrimidine quinoline tetracaine HCl

a) b)

MMEEKC log k’ 0.37 ± 0.00 0.68 ± 0.01 -0.43 ± 0.01 -0.14 ± 0.01 -0.20 ± 0.01 -0.61 ± 0.01 0.60 ± 0.03 0.06 ± 0.01 0.20 ± 0.01 0.40 ± 0.00 0.34 ± 0.01 0.94 ± 0.02 1.24 ± 0.07 0.34 ± 0.00 0.39 ± 0.03 0.86 ± 0.00 1.33 ± 0.03 1.10 ± 0.01 0.15 ± 0.00 0.38 ± 0.02 0.62 ± 0.03 0.17 ± 0.01 1.17 ± 0.01 -0.98 ± 0.01 -1.08 ± 0.01 0.50 ± 0.00 1.39 ± 0.02

MMEEKC log POW 1.80 ± 0.01 2.33 ± 0.02 0.42 ± 0.01 0.91 ± 0.02 0.82 ± 0.01 0.11 ± 0.01 2.20 ± 0.05 1.26 ± 0.01 1.51 ± 0.01 1.84 ± 0.01 1.74 ± 0.02 2.78 ± 0.03 3.30 ± 0.11 1.74 ± 0.01 1.82 ± 0.06 2.64 ± 0.01 3.45 ± 0.05 3.06 ± 0.03 1.42 ± 0.01 1.82 ± 0.04 2.23 ± 0.05 1.45 ± 0.01 3.18 ± 0.01 -0.53 ± 0.02 -0.71 ± 0.02 2.03 ± 0.01 3.56 ± 0.04

Literature b log POW 1.71 2.25 0.49 0.90 0.64 -0.07 1.88 12 0.85 1.39 1.96 1.86 2.64 13 3.43 1.35 1.77 2.26 3.30 3.0513 1.17 1.85 1.91 1.49 3.20 -0.23 -0.40 2.03 3.73

∆log POW 0.09 0.08 -0.08 0.01 0.18 0.18 0.32 0.41 0.12 -0.12 -0.12 0.14 -0.13 0.39 0.05 0.38 0.15 0.01 0.25 -0.03 0.32 -0.04 -0.02 -0.30 -0.31 0.00 -0.17

Average of 4 replicates ± stdev in a single run. Values were obtained from reference 11 unless otherwise noted.

Table 2. MMEEKC log k’ values and MMEEKC log Pow values determined with the MCE 2000™.

Summary The ability of the MCE 2000™ system to conduct high throughput estimations of log Pow values by MMEEKC has been demonstrated. The MMEEKC log Pow values obtained from the MCE 2000™ were very close to the established literature values. The use of a 96-capillary array allows for the simultaneous determination of up to 23 different compounds along with a standard mixture in quadruplicate in a single CE run, a substantial improvement in throughput over single capillary approaches. The flexibility of the MCE 2000™ system provides the ability to also conduct high throughput pKa14 screening with the same instrument simply by switching out capillary arrays and buffer solutions. This unique ability results in a cost effective, high throughput approach for characterizing drug candidate physicochemical properties.

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References (12) (1) (2)

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Leo, A.; Hansch, C.; Elkins, D. Chem. Rev. 1971, 71, 525. Ishihama, Y.; Oda, Y.; Uchikawa, K.; Asakawa, N. Anal. Chem. 1995, 67, 1588-1595. Poole, S. K.; Durham, D.; Kibbey, C. J. Chromatogr. B 2000, 745, 117125. Gluck, S. J.; Benkö, M. H.; Hallberg, R. K.; Steele, K. P. J. Chromatogr. A 1996, 744, 141-146.

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Kibbey, C. E.; Poole, S. K.; Robinson, B.; Jackson, J. D.; Durham, D. J. Pharm. Sci. 2001, 90, 1164-1175. Klotz, W. L.; Schure, M. R.; Foley, J. P. J. Chromatogr. A 2001, 930, 145154. Watarai, H. Chem. Lett. 1991, 391394. Wehmeyer, K. R.; Tu, J.; Jin, Y.; King, S.; Stella, M.; Stanton, D.; Strasburg, R.; Kenseth, J.; Wong, K.S. Submitted for publication in LCGC. Application note #APP-207500, CombiSep, Inc.: Ames, IA, 2002. Wong, K.-S.; Kenseth, J.; Strasburg, R. Submitted for publication in J. Pharm. Sci. Hansch, C.; Leo, A.; Hoekman, D. Exploring QSAR [2]. Hydrophobic, electronic, and steric constants; American Chemical Society: Washington, DC, 1995. Berthod, A.; Carda-Broch, S.; Garcia-Alvarez-Coque, M. C. Anal. Chem. 1999, 71, 879-888. SRC's LOGKOW/KOWWIN Program: http://esc.syrres.com/interkow/kowde mo.htmT. Application note #APP-111200, CombiSep, Inc.: Ames, IA, 2002.

Kit-Sum Wong is an application scientist and Jeremy Kenseth is an application scientist and laboratory manager at CombiSep, Inc., Ames, IA.

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