A Rapid Method for pKa Determination of Drugs Using Pressure-Assisted Capillary Electrophoresis with Photodiode Array Detection in Drug Discovery

A Rapid Method for pKa Determination of Drugs Using Pressure-Assisted Capillary Electrophoresis with Photodiode Array Detection in Drug Discovery YASUSHI ISHIHAMA, MASAHIRO NAKAMURA, TOSHINOBU MIWA, TAKASHI KAJIMA, NAOKI ASAKAWA Analytical Research Laboratories, Eisai Co., Ltd., 5-1-3 Tokodai, Tsukuba, Ibaraki 300-2635, Japan Received 17 April 2001; revised 21 November 2001; accepted 21 November 2001

ABSTRACT: We developed a rapid screening method for determination of pKa of candidate drugs by pressure-assisted capillary electrophoresis (CE) coupled with a photodiode array detector. Application of pressure during CE analysis allowed completion of one CE run in less than 1 min, and the obtained pH-metric mobility shifts as well as pH-metric UV spectrum were analyzed by a nonlinear regression ®tting software to determine pKa values. The difference between pKa values by this method and by other conventional methods is within 0.25 units for 82 ionic functional groups of 77 drugs. The pKa values of 96 compounds in dimethylsulfoxide solution on a 96-well microplate could be measured in 1 day. Our method provides rapid and accurate determination of pKa value.

ß 2002 Wiley-Liss, Inc. and the American Pharmaceutical Association J Pharm Sci 91:933±942, 2002

Keywords: detection

pressure-assisted capillary electrophoresis; pKa; HTS; photodiode array

INTRODUCTION Ionization of drugs is important because it affects not only the physico-chemical properties of the drugs such as lipophilicity and solubility, but also several parameters related to cell±drug interaction such as membrane permeability, plasma protein binding, metabolism, tissue penetration, and target protein binding. The acid dissociation constant (pKa) is an index of the extent of ionization of a drug at different pH values, and is therefore, an important parameter that re¯ects optimization of the drug structure. Recent advances in combinatorial technology have enhanced the production of candidate drugs as well as the discovery of new drugs. The highthroughput screening (HTS) method is used for determination of drug bioactivity as well as the physicochemical properties of drugs such as lipoCorrespondence to: Yasushi Ishihama (Telephone: ‡45 6550 2365; Fax: ‡45 6593 3018; E-mail: [email protected]) Journal of Pharmaceutical Sciences, Vol. 91, 933±942 (2002) ß 2002 Wiley-Liss, Inc. and the American Pharmaceutical Association

philicity,1,2 solubility,3±6 and membrane permeability,7±9 and allows evaluation of the increasing number of candidate drugs. To our knowledge, however, there are no methods suitable for HTS pKa determination. For any pKa method to be useful, it should have the following characteristics. (1) The daily throughput should be more than 96 compounds (1 microplate) because the compound libraries are managed on the basis of the microplate unit at present. (2) The required amount of the sample should be less than 1 mg. (3) Samples with unknown purity and stability should be applicable. (4) The sensitivity of pKa measurement should be independent of the sample structure. Traditionally, potentiometric titration, spectrophotometry, solubility, and liquid±liquid partitioning have been used for determination of pKa. Among these, the solubility and partitioning methods are time consuming for measurement of pKa in the high throughput mode. While the automated titration method is also slow, titration with organic solvents is effective for sparing soluble drugs.10 The most promising method is

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spectrophotometry, because it is rapid and sensitive to sparing soluble drugs. However, its major disadvantage is that it cannot be applied to compounds lacking chromophores close to ionophores. Although the modi®ed instrument has been reported to improve the sensitivity,11 the difference in the sensitivity between compounds with and without pH-sensitive chromophores is problematic when spectrophotometry is applied to a wider range of compounds including sparingly soluble drugs and multivalent ionic drugs. In addition, all the above methods are affected by the purity/stability of the samples. Other chromatographic approaches such as HPLC are suitable for the samples with low purity/stability.12 However, it is dif®cult to apply the single HPLC condition to a wide range of compounds with varied hydrophobicity. On the other hand, computation of pKa by using appropriate software is quite useful if the accuracy is well validated for a wide range of compounds. Unfortunately, however, the computed values of pKa are often inaccurate at present. For example, the Hammett-Taft method cannot provide reliable values especially for novel structures because it requires the substituent constants of the partial structures.13 pKa can also be determined by the electrophoresis method.14 Since the description of this method, pH-metric electrophoretic mobility-shift has been used for samples with low purity/ stability.15±17 The automated capillary electrophoresis (CE) instrument in combination with a general equation for multivalent compounds allows rapid and accurate pKa determination for multivalent drugs.18 The most attractive feature of this technique as an HTS method is its universal applicability to a wide range of compounds, i.e., the mobility always shifts when the extent of ionization of the analyte is modi®ed because the mobility is proportional to the net charge. Therefore, unlike the spectrophotometric method, the applicability of CE method is independent of the structural diversity of the analytes. In addition, the detection limit of pKa by capillary electrophoresis with an ultraviolet (UV) detector is approximately 10 6 M.17,18 However, the CE method has one disadvantage; measurements under acidic conditions are quite time consuming and less reproducible because of the suppression of the electroosmotic ¯ow (EOF) inside the fused silica capillary.18 Although the use of an anionic polymer-coated capillary has been reported to be effective in overcoming this shortfall,19,20 the JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 91, NO. 4, APRIL 2002

analysis time remained unsatisfactory so as to perform rapid screening with daily output of 96 samples on the microtiter plate. Recently, two different groups reported the faster CE methods for pKa determination using assistance of pressure.21,22 However, the throughput was still unsatisfactory for 96-well microplate analysis. In the present report, we demonstrated the analysis of 96 compounds in 1 day using pressureassisted CE with a photodiode array (PDA). We also discuss the applicability of this rapid measurement system on drug discovery.

EXPERIMENTAL All experiments for pKa measurement were conducted using a Beckman-Coulter P/ACE MDQ capillary electrophoresis (Fullerton, CA) with a photodiode array detector (200±400 nm). Untreated fused silica capillaries with 50 mm i.d. and 31-cm length were obtained from GL Sciences (Tokyo, Japan). Fifty microliter each of sample stock solutions of maximum 96 compounds in dimethylsulfoxide (DMSO, 10±30 mM) was prepared in a 96-well microplate (Nalge-nunc, Tokyo). Then, 10 mL of each solution was transferred to each well of a 96-well sample tray of the CE instrument and was 20-fold diluted by water. The transfer and dilution steps were automatically done using Biotec EDR-500A auto dropper (Tokyo) in less than 1 min. Separation buffers employed were phosphate, acetate, or borate with ionic strength ˆ 0.05, as described previously.18 In this study, three sets of buffers such as two sets of six pH buffers (pH 3±8, pH 6±11) and one set of nine pH buffers (pH 3±11) were prepared. Among them, one set was selected according to the structure of the analyte. Applied voltage was 10 kV. Pressure at 1.5 psi was applied at the anode vial using the pressure function of the instrument. The capillary was thermostated at 258C by a liquid coolant. The time program of the method consisted of six or nine cycles of three steps such as buffer-rinsing step (100 psi, 15 s), injection step (0.5 psi, 10 s), and separation step (0.9 or 1.5 min). Obtained migration times of DMSO and drugs at appropriate wavelength were converted to effective mobilities as follows;   1 1 Ll meff ˆ  …tm a…n 1†† …t0 a…n 1†† V …1†

RAPID pKa SCREENING BY CAPILLARY ELECTROPHORESIS

where meff is the effective mobility, tm is the migration time of the drug, t0 is the migration time of DMSO, L is the total length of the capillary, l is the length to the detector, V is the applied voltage, a is the time for one cycle and n is the number of the cycles. Then, an MS-Excel (Microsoft, Redmond, WA) nonlinear ®tting program based on Newton method was run to obtain the pKa values of a general multivalent compound, BHn i Ai or BHn‡j Aj‡ (0  i  n, 0  j  m), using the following equation as described previously:18 ! n i m b fH ‡ gj Q P P ai j 0 f …n† K ‡ f …m† j ak Q ‡ i 0 iˆ1 fH g kˆ1 jˆ1 Kbl lˆ1 meff ˆ 0Q 1 0 1 i 0 K j ‡ n m ak P B fH g C PB C f …n† @kˆ1 ‡ i A ‡ 1 ‡ f …m† @ Q A j 0 iˆ1 fH g jˆ1 Kbl lˆ1

…2†

0 0 where Kai , Kbj , and f(x) are given by   fH ‡ g BHn i Ai 0 Kai ˆ ‰BHn i‡1 A…i 1† Š

0 Kbj

 fH ‡ g BHn‡j 1 A… j   ˆ BHn‡j A j‡

1†

…3†

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sodium, and E-No compounds, which were obtained from Eisai Kashima Plant (Hasaki, Ibaraki, Japan). All other reagents were of analytical grade. All buffer solutions were ®ltered through a membrane ®lter of 0.45-mm pore size (Chromatodisk, GL Sciences).

RESULTS AND DISCUSSION Figure 1A shows the structures of donepezil, an acetylcholinesterase inhibitor for Alzheimer's disease,25 and its analogs. The measured pKa values were 9.1 for donepezil, 9.0 for 1, and 7.2 for 2, while a constant value of 8.72 was computed for all the compounds by the calculation program when the most popular Hammett-Taft method was applied.13,26 The same tendency was obtained for monobasic b-blockers (Figure 1B) as well as piperazine derivatives developing in our laboratories (Figure 1C). In the latter case, pKa values affect the bioavailability because these values are very close to the pH in the intestinal portion

 …4†

f …x† ˆ 0 …x ˆ 0†; f …x† ˆ 1 …x 6ˆ 0†

…5†

and ai(> 0) and b j are de®ned as the mobility of BHn i Ai and BHn‡j Aj‡, respectively, at an ionic strength. Note that ai is not always equal to i a1 (e.g., betahistine, see Figure 8). To obtain thermodynamic pKa (pKth a ), the Debye-Huckel equation was used for drugs except zwitterions, whose activity coef®cients could be obtained not from the Debye-Huckel equation but by other techniques such as osmotic pressure measurement.23 In this study, therefore, pK0a (I ˆ 0.05) was used as pKa for zwitterions, while pKth a was employed as pKa for other drugs, as described previously.18 Regarding the UV method for betahistine, an MS-Excel nonlinear ®tting program based on the Newton method was run to obtain the pKa values using the following equation: Sˆ

SBH  10 10

pKa pKa

‡ SBH2  10 ‡ 10 pH

pH

…6†

where SAH and SAH2 are the absorbance of BH‡ 24 and BH2‡ 2 at 270 nm, respectively. All drugs employed were from Sigma (St. Louis, MO) except donepezil hydrochloride, rabeprazole

Figure 1. Structures of donepezil and its analogues, b-blockers, and piperazine derivatives. JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 91, NO. 4, APRIL 2002

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where these drugs are absorbed. Therefore, it is quite important to determine pKa values not by calculation programs but by actual measurement. In general, the daily throughput of the normal CE method is less than 20 compounds. To increase the total throughput, we applied air pressure to the capillary end with voltage during CE analysis. In addition, to accelerate the sample preparation time, diluted solutions of 10 mM compound stock solutions in DMSO were used as the sample solutions because the stock solutions used for biological assay were usually prepared prior to pKa analysis and DMSO works as the EOF marker instead of mesityl oxide. In this pressure-assisted mode, however, the parabolic ¯ow pattern by pressure disturbed the ¯at ¯ow of EOF caused by electrical ®eld, as expected. Consequently, at 1.5 psi, the theoretical plate number of drugs was markedly decreased to approximately 10%, whereas the ¯ow rate of the EOF marker increased threefold and the analysis time was reduced to less than 1 minute (Figure 2). Also we simulated the ionization percentage of donepezil required for the resolution (Rs) equal to 0.5 between peaks of donepezil an DMSO using the theoretical

plate, the mobility of DMSO with pressure and voltage, and the mobility of donepezil with 100% ionization. As shown in Figure 2B, at more than 1.5 psi, separation was incomplete at pHs where the ionization is less than 50%, and the accuracy of this method would be decreased. To compensate for the decrease in the ef®ciency of the time axis, two-dimensional analysis using PDA detector was performed. DMSO has little absorbance over 250 nm, and thus, it was relatively easy to separate the solute peak from that of DMSO when drugs have chromophores at more than 250 nm. Typical electropherograms of donepezil (pKa 9.1) at different wavelengths at pH 9 are shown in Figure 3. Although the separation between DMSO and donepezil was not accomplished at 220 nm as expected, the separation was successfully done at 246 or 280 nm. Thus, the migration time of donepezil was measured at 246 or 280 nm, whereas the migration time of DMSO was measured at 220 nm. In addition, at 1.5 psi, analyses were ®nished in 0.9 min even when acidic conditions were employed. Next, we modi®ed the time program of the separation method. Usually, the method consists of a rinsing step with the buffer, injection step, and the separation step with application of voltage and pressure. The procedure is repeated using buffers of six or nine different pH values. Therefore, six or nine electropherograms are analyzed

Figure 2. In¯uence of applied pressure on CE separation of donepezil. Conditions: separation solution, phosphate (I ˆ 0.05, pH 7); sample, 20-fold dilution solution of 10 mM donepezil in DMSO by water; other conditions as described in the Experimental section. (A) Dependence of the migration time and the mobility of DMSO on applied pressure. (B) Dependence of theoretical plate of donepezil and ionization percentage of donepezil required for Rs ˆ 0.5 between DMSO and donepezil on applied pressure. Ionization percentage, x, was calculated from theoretical plate, N, at certain pressure using the following equations: p 1 meff   N ˆ 0:5; m 4 eff ‡ m eof 2 x x pH7 ˆ 1:4  10 8  ˆ meff  100 100

Rs ˆ meff

where meff and meof are the effective mobility of donepezil and the apparent mobility of DMSO, respectively. JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 91, NO. 4, APRIL 2002

Figure 3. Electropherograms of donepezil at different wavelengths. Conditions: separation solution, borate (I ˆ 0.05, pH 9); others are described in the Experimental section.

RAPID pKa SCREENING BY CAPILLARY ELECTROPHORESIS

to derive the pKa value of the drug. In this study, we bundled the six or nine assays into one single procedure consisting of six or nine cycles of the above three steps using different buffers, and obtained the pKa value from the single electropherogram. The above protocol also signi®cantly reduced the run time (one sixth-fold decrease) by reducing the instrument calibration time, which requires 2 min per method. Accordingly, the time required for analysis of one sample decreased to 14 min, i.e., more than 100 samples per 24 h, even when nine pH buffers were employed. A typical example of the established method for donepezil is shown in Figure 4. The observed pH-metric mobility curve was ®tted using nonlinear regression analysis based on Gauss-

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Newton method (Figure 5A). Also, the pH-mobility curve of a monoacidic drug, sulfameter, is shown in Figure 5B. Note that separation between DMSO and the drug was successful at pH 5, where the negative charge of sulfameter is 0.03, i.e., the ionization is 3%. In the next step, we compared our rapid CE method with other conventional methods. For this purpose, we evaluated 77 drugs including 82 acidic and basic functional groups. Our results showed a high agreement between rapid CE method and conventional methods from pKa 2.4 to 10.9 (Figure 6). The details are described in Table 1. The difference was from 0.25 to ‡0.20 unit, and the 95% con®dential range of the difference was within 0.20 unit.

Figure 4. Electropherograms of donepezil by the rapid CE method. Conditions are described in the Experimental section. JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 91, NO. 4, APRIL 2002

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Figure 5. Dependence of effective mobility of (A) donepezil and (B) sulfameter on pH. Conditions are described in the Experimental section.

The repeatability of this method was investigated for donepezil, 4-nitrophenol and ketoprofen (Table 2). Although the RSD values of the mobilities at pHs near their pKa values were over 3%, the repeatability of their pKa values was satisfactory, and the RSD were less than 1%. This was caused by the characteristic of the sigmoidal curve ®tting.27 These results also indicate that the buffer sets employed allow the 20 times analyses without exchange. This rapid CE method was useful for analysis of drugs with low stability such as rabeprazole, which is quite unstable under acidic conditions.

Figure 6. Relationship between pKa values by rapid CE method and by other methods for 82 ionic functional groups of 77 drugs. JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 91, NO. 4, APRIL 2002

In this regard, the normal CE method could not be applied at pH below 5.0 because of the complete degradation within 5 min. On the other hand, analysis in less than 1 min at every pH could be conducted by the rapid CE method. Figure 7 shows that measurement of pKa of the pyridinium group was possible when our rapid method was applied. Another example was the identi®cation of the ionic group of multivalent species by combining our rapid protocol with the pH-metric UV method by using a PDA detector. Betahistine, N-methyl-2-pyridineethanamine, has two ionic groups, secondary alkylamine and pyridine, in its structure. We observed two sigmoidal changes in the pH-metric mobility curve by the rapid CE method, whereas one sigmoidal change was observed in the pH-metric UV curve at 270 nm (Figure 8). Considering the sensitivity of the pHmetric chromophore, the pKa values of the pyridinium group and alkylammonium group were 5.21 and 10.13, respectively. In the drug discovery stage, it is more important to identify ionic groups with close pKa values of amphoteric candidates. In particular, determination of whether the existing species are the neutral or zwitterionic forms is quite important because the characteristics of these forms are quite different such as solubility and absorption. For example, we analyzed two candidates that had the same phenol group and different guanidinium groups, and close pKa values. They showed different cell membrane permeability and bioavailability, i.e., one was more than 10% while the other was less than 1%. We measured these two compounds by our rapid CE/PDA system and found that under physiological conditions, one was a mixture of nonionic and cationic forms, while the other was a mixture of the zwitterionic and cationic forms. The results of such analysis were quite important for estimating the difference in intestinal permeability. The limitation of this method is the application to drugs without UV chromophore at more than 250 nm. In some cases, it was effective to remove DMSO by evaporation under vacuum followed by the addition of methanol or acetonitrile as a neutral marker. Also, the drugs with solubility lower than 0.1 mg/mL could not be analyzed by this method at present. In such cases, we added some organic solvents to the separation buffers and extrapolated to 100% aqueous solutions although the throughput was decreased. In conclusion, we developed a rapid method for pKa determination of more than 96 candidate

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Table 1. pKa Values of 77 Compounds by Rapid CE and Other Methods pKa No.

Compound

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53

Tryptophan I Enalapril Salicylic acid E1101 Aspirin Furosemide Diclofenac Flurbiprofen E3330 Adenine I Sulindac Ketoprofen Flufenamic acid Naproxen Ibuprofen Phenylbutazone Pravastatin Fluvastatin Indomethacin Rabeprazole S-oxide I Warfarin Rabeprazole I Lamotrigine Betahistine I Sulfamethizole Piroxicam Sulfachloropyridazine Compound 4 Sulfadimethoxine Sulfameter Cimetidine Compound 3 4-Nitrophenol Cephalexin Sulfathiazole Scopolamine Compound 2 4-Hydroxybenzaldehyde Rabeprazole S-oxide II Diltiazem Sulfamethazine 4-Cyanophenol 40 -Hydroxyacetophenone Clonidine Pentobarbital Phenytoin Terbutaline 4-Hydroxybenzamide Verapamil Theophilline Rabeprazole II Compound 1 Sulpiride

This Method

Other Methods

Difference

2.38 3.00 3.08 3.28 3.47 3.58 3.99 3.91 4.13 4.18 4.08 4.14 4.10 4.20 4.27 4.33 4.36 4.39 4.51 4.70 4.98 5.13 5.34 5.21 5.24 5.27 5.58 5.92 5.99 6.47 6.68 6.72 7.06 7.06 7.19 7.40 7.15 7.45 7.63 7.72 7.80 7.83 7.92 8.14 8.17 8.18 8.23 8.33 8.60 8.66 8.67 8.99 8.99

2.38 3 3 3.26 3.49 3.52 4 3.8 4.10 4.17 4.13 4.29 3.9 4.15 4.31 4.5 4.2 4.32 4.5 4.73 5.03 5.18 5.5 5.26 5.45 5.1 5.5 5.94 6.2 about 6.8 6.8 6.71 7.15 7.14 7.2 7.53 7.2 7.62 7.69 7.7 7.70 7.77 8.05 8.05 8.18 8.21 8.31 8.18 8.59 8.77 8.64 9.00 9

0 0 0.08 0.02 0.02 0.06 0.01 0.11 0.03 0.01 0.05 0.15 0.2 0.05 0.04 0.17 0.16 0.07 0.01 0.03 0.05 0.05 0.16 0.05 0.21 0.17 0.08 0.02 0.21 0.25 0.12 0.01 0.09 0.08 0.01 0.13 0.05 0.17 0.06 0.02 0.10 0.06 0.13 0.09 0.01 0.03 0.08 0.15 0.01 0.11 0.03 0.01 0.01

References Ref. 28 Ref. 28 Ref. 29 Ref. 18 Ref. 29 Ref. 30 Ref. 28 Ref. 29 Ref. 18 Ref. 18 a

Ref. 30 Ref. 30 Ref. 29 Ref. 30 Ref. 29 Ref. 31 Ref. 26 Ref. 28 b

Ref. 18

b,c

Ref. 32

a

Ref. 28 Ref. 29 Ref. 33 b

Ref. 33 Ref. 28 Ref. 30

b

Ref. 34 Ref. 35 Ref. 33 Ref. 36

a a b

Ref. 30 b

a b

Ref. 37 Ref. 30 Ref. 30 b

a

Ref. 38 Ref. 28 b

a

Ref. 28

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Table 1. (Continued ) pKa No.

Compound

54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82

Nadolol Donepezil (E2020) 4-Bromophenol 4-Iodophenol 4-Hydroxybiphenyl Chlorpheniramine Tryptophan II Acetaminophen Propranolol Oxprenolol Metoprolol Alprenolol Acebutolol Atenolol 4-Fluorophenol Phenol Adenine II E3620 4-Methoxyphenol 4-Ethylphenol 4-Methylphenol 4-nButoxyphenol Betahistine II 4-Ethoxyphenol 4-tButylphenol Oxymetazoline Naphazoline Tetrahydrozoline Xylometazoline

This Method

Other Methods

Difference

9.15 9.08 9.20 9.21 9.33 9.36 9.40 9.46 9.49 9.49 9.51 9.52 9.56 9.57 9.94 9.78 9.77 9.95 10.21 10.00 10.08 10.11 10.13 10.14 10.14 10.61 10.65 10.79 10.91

9.23 9.1 9.34 9.2 9.38 9.2 9.39 9.7 9.49 9.5 9.56 9.7 9.67 9.55 9.82 9.99 9.75 9.95 10.18 10.00 10.26 10.26 10.14 10.25 10.23 10.51 10.61 10.88 11.02 Average Max Min SD

0.08 0.02 0.14 0.01 0.05 0.16 0.01 0.24 0 0.01 0.05 0.18 0.11 0.02 0.12 0.21 0.02 0 0.03 0 0.18 0.15 0.01 0.11 0.09 0.10 0.04 0.09 0.11 0.03 ‡0.20 0.25 0.099

References b

Ref. 18 Ref. 34 Ref. 34 a b

Ref. Ref. Ref. Ref. Ref. Ref. Ref. Ref. a

28 39 38 37 30 37 37 37

Ref. 34 Ref. 18 Ref. 18 b

Ref. 34 Ref. 34 Ref. 30 b

Ref. Ref. Ref. Ref. b

30 34 38 38

b

a

By spectrophotometry according to ref. 24. By conventional CE method according to ref. 18. c Extrapolated value using data above pH 5. b

Table 2. Repeatability of Measurement (n ˆ 20) RSD (%)a Compound Donepezil 4-Nitrophenol Ketoprofen

Migration Effective Time Mobility pKa  SD (RSD%) 1.29 1.56 3.30

6.60 4.29 3.63

9.08  0.06 (0.64%) 7.06  0.03 (0.49%) 4.14  0.02 (0.57%)

a RSD (%) values for the migration time and the effective mobility were calculated from the results at pH 9 for donepezil, pH 7 for 4-nitrophenol and pH 4 for ketoprofen.

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Figure 7. pKa determination of rabeprazole by the rapid CE method. Conditions are described in the Experimental section.

RAPID pKa SCREENING BY CAPILLARY ELECTROPHORESIS

Figure 8. pKa determination of betahistine by mobility and UV methods. Conditions are described in the Experimental section.

drugs in 1 day that allows selection of appropriate drug or the design of optimal structure. Our pressure-assisted CE with PDA system is a powerful tool in the stage of lead optimization to the design of the candidate drugs as well as the physico-chemical characterization of the compound libraries.

ACKNOWLEDGMENTS The authors would like to thank Yasuyuki Suzuki for his technical assistance as well as helpful suggestions.

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