A Strategy for Performing Rapid pKa Measurements on Insoluble Compounds by Multiplexed 96-Capillary Electrophoresis Jeremy Kenseth, Andrea Bastin, and Dennis Tallman CombiSep, Inc., 2711 South Loop Dr., Suite 4200, Ames, IA 50010
Instrumentation cePRO 9600™ 96-Capillary Multiplexed CE-UV System
• 96-capillary array CE instrument with fixed wavelength UV detection • Robotic interfacing capabilities
Experimental • Different acidic, basic, and zwitterionic compounds with literature pKa values ranging from 10 and with a wide range of aqueous solubilities were selected for the study. • 100 ppm (0.1 mg/ml) solutions of analytes were prepared in 0.1 % DMSO (EOF neutral marker). Basic compounds were dissolved in 1 mM H3PO4 and acidic compounds were dissolved in 1 mM NaOH. Some sample solutions also contained up to 20% methanol to enhance compound solubility. • A 24-component buffer series (pH range from 1.7 - 11.2; I = 50 mM) was used for collecting the electrophoretic data. A 12-component buffer system (pH range 2.1-10.8) was also used. • Prior to each CE run, the capillary array was flushed for 2 min at 40 psi with 10 mM sodium tetraborate buffer (pH 9.1). The various pH buffers were loaded into a 96-well plate and vacuum injected into the capillary array at –2.0 psi for 2-5 min before sample injection. • Optimized CE conditions (33 cm effective/55 cm total length capillary array): -0.2 psi, 5 sec sample injection; 3.5 kV, -0.2 psi vacuum-assisted CE; 13 min run time. • Data Analysis: Apparent pKa values at I = 50 mM (pKa’ values) were determined using pKa Estimator™ software (CombiSep, Inc., Ames, IA). The effective mobility (meff) was determined at each pH value from the migration time difference between the analyte and DMSO. Non-linear regression was performed on the data, estimating the analytes’ pKa’ value(s). • Prediction of compound octanol-water partition coefficients (ClogP) and aqueous solubilities (CSol) were performed using ACD/Labs Online (I-Lab) V 7 software (Advanced Chemistry Development, Inc. (ACD/Labs)). Only predictions for compounds with CSol below 100 ppm are listed.
Attributes of Multiplexed CE-UV for pKa Analysis High Sample Throughput: Up to 24 compounds/h can be analyzed for pKa when using 12 pH values; 12 compounds/h can be analyzed when using 24 pH values.
Aqueous pKa Measurements by Multiplexed Capillary Electrophoresis Compound
MW
# Runs
cePRO pKa' (IS = 50 mM)
SD
Literature pKa Values
References
CpKa
Acebutolol Acetaminophen Acetylsalicylic Acid Acyclovir
336 151 180 225
10 16 21 12
250 137
10 9
2-Aminopyridine 4-Aminopyridine Atenolol Benzoic Acid Benzylamine Betahistine
94 94 266 122 108 136
38 97 12 58 126 20
Bifonazole Cefadroxil
310 363
3 16
Cefuroxime
423
3
Cephalexin
329
3
9.55; 9.56 9.56; 9.46; 9.75; 9.45 3.30; 3.47; 3.51 2.25; 2.16 9.31; 9.31 9.55; 9.52 2.45 4.99 6.70 9.18; 9.02 9.56; 9.57; 9.64; 9.52; 9.43; 9.61 3.98; 4.12; 4.26; 4.22 9.71 5.21; 3.46 10.13; 9.78 5.72 2.47; 2.86 7.41; 7.14 9.89; N.R. 2.04 N.R. ppt; N.R.; 3.11; 2.53 ppt; 7.06; 6.79; 7.14
Chloroamphetamine Chloroquine
170 320
16 26
0.02 0.06 0.04 0.04 0.03 0.03 0.08 0.02 0.07 0.05 0.04 0.05 0.07 0.13 0.04 0.01 0.08 0.08 0.09 0.01 0.13 0.02 0.02 0.10 0.11 0.04
A,B A,B,C,D A,B,E A,E
Alprenolol 4-Aminobenzoic Acid
Chlorpromazine* Chlorthalidone
319 339
3 3
Cimetidine Clozapine
252 327
16 16
Clomipramine* Clotrimazole* Codeine Desipramine Diethylstilbestrol
315 345 299 266 268
8 12 10 12 4
9.48 9.38 3.48 2.18 9.18 9.55 2.28 4.77 6.71 9.22 9.56 4.09 9.54 4.00 10.00 6.22 2.59 6.96 9.59 2.14 11.16 2.51 7.09 9.85 7.11 10.57 Cosolvent Required 9.13 11.08 6.80 3.85 7.52 9.54 6.03 8.09 10.42 9.78 >11.2 3.87 6.33 3.59 10.33 5.73 9.87 4.06 9.13 4.31 9.61 3.90
0.04 0.01 0.01 0.01 0.01 0.06 0.08 0.08 0.01 0.05 0.01
3.63 6.32; 6.42; 6.44; 6.38; 6.31 3.35; 3.58; 3.64; 3.65; 3.74 10.15; N.R.; 10.6; 10.24; 10.90 N.D.
4.01; 4.27; 4.24; 4.29; 4.51 ppt; 9.66; 9.58; 9.13 ppt; 4.06
A,B,D,E,H A,C,Q,R A,C
9.11 9.86 3.48 2.01 9.35 9.17 2.51 4.86 6.67 9.26 9.17 4.20 9.40 3.45 9.75 6.14 N.D. N.D. N.D. 2.59 8.76 3.12 6.78 10.07 6.33 10.49 9.41 8.57 10.85 7.12 5.67 7.14 9.46 5.33 8.25 10.42 10.06 10.74 3.67 5.72 3.04 9.79 5.45 10.15 4.77 10.10 4.41 9.49 3.96
7.31 9.10 1.18 3.63 5.23 10.41 7.92 10.29 8.75 9.91 9.44 9.56 2.47 Cosolvent Required 7.93 9.30 9.08 4.30 8.46 3.30 8.12 1.85 4.73 2.17 8.17 6.92 9.06 10.23 6.26 2.64 2.20 9.04 8.17 1.76 5.39 6.95 2.09 9.09 9.14 9.61 7.12 Cosolvent Required 4.14 8.47 2.80 8.41 9.31 1.22 5.33 Cosolvent Required 2.13 8.52 6.78
0.05 0.04 0.19 0.02 0.03 0.04 0.04 0.09 0.03 0.07 0.04 0.03 0.00
7.42; 7.41 9.38; 9.36 N.R. 3.10 4.80 10.40 7.83; 7.96; 7.92 10.20 - 10.44 9.61 9.80 9.42; 9.48 9.58; 9.51 2.38 ppt; 6.63 8.23 9.32
A,D
Flufenamic Acid Flumequine Furosemide
281 261 331
3 12 2
Histamine
111
3
Hydroquinine
326
16
Ibuprofen Imipramine Indomethacin
206 280 358
12 8 3
Labetalol
229
16
Leucovorin
471
3
Lidocaine Maprotiline Metaproterenol
234 277 211
12 16 10
2-Methylbenzylamine Metoprolol Metronidazole Miconazole* Morphine
121 320 171 416 285
15 12 3 7 10
Nafronyl Naproxen Nefopam Nicotine
384 230 253 162
10 6 10 23
Nicotinic Acid
123
6
Nifedipine 3-Nitrophenol 4-Nitrophenol Norephedrine Nortriptyline Papaverine Penicillin-G Phenylalanine
346.3 139 139 151 263 339 333 165
3 38 38 3 9 38 8 4
Physostigmine Piroxicam
275 331
3 6
Prazosin Procaine
385 300
12 18
Promethazine Propanolol Quinacrine*
284 260 400
9 10 8
Quinine
324
42
Salicylic Acid Sotalol
138 272
23 10
Sulfacetamide
214
2
Small Sample Consumption: Approximately 5-10 µg per sample well required for analysis; typical sample volume of 50-100 µl; only nl volumes injected Separation of Potential Interferents: As CE is a separation technique, sample impurities or degradants possessing different charge-to-mass ratios can be resolved from target compound Sample Requirements: Only UV absorptivity at 214 nm required for detection; spectral changes between ionization states of compounds not required; exact concentration of sample does not need to be known
Tamoxifen* Tetracaine
371 264
3 10
Trazodone
372
16
Trimipramine* Tryptophan
295 204
8 4
Tyrosine
181
4
Warfarin
Multiplexed Format: Provides the capability to effectively span a wide pH range (pH 1.8 to pH 11.2) with good resolution (0.4 pH units) to identify extreme pKa values, deconvolute closely spaced pKa values, and improve confidence in results
Average
N.R. ppt * #
308
12
9.48 2.30 9.25 2.21 8.82 10.01 4.94
0.02 0.16 0.06 0.08 0.07 0.05 0.05 0.03 0.04 0.03
0.03 0.04 0.04 0.01 0.03 0.11 0.07 0.03 0.02 0.01 0.04 0.04 0.06 0.03 0.07 0.04 0.01 0.00 0.06 0.03 0.02 0.05 0.10 0.04 0.06 0.05 0.09 0.05 0.07 0.05 0.06 0.05 0.06 0.01 0.09 0.06 0.05 0.03 0.00 0.01 0.01 0.02 0.03 0.08
A,B F E E,G A,B,C,E,G,H A,E,G,H H B,I J E,K
K A,B,K,L M M
9.24; 9.16 9.11 10.98 6.68; 6.89; 6.97 N.R. 7.63 9.40 6.54 8.21; 7.97 10.17
L,N A B,C,E O P M C,H A # Q A,C,D,E,G A,B,C,D,E A M
4.2; 4.13 9.36 3.13; 3.25; 3.00; 3.10 8.24; 8.13; 8.29; 8.02 2.43; N.R.; 2.10: N.R. 4.62: 4.75; 4.63; 4.84 -1.83 8.33 7.06; 7.05; 6.93 10.43; 9.7; 10.14 6.45; 6.25; 6.47; 6.21; 6.38 3.76; 2.7 1.83 9.12 8.13; 8.17 2.53; N.R. 5.32; 5.27 7.04 2.27 9.15 9.1; 8.62 9.60; 9.49; 9.57; 9.61; 9.49 7.73 10.20 4.22; 3.95; 4.33; 4.18; 4.13; 4.14 8.55; 8.6; 8.59; 8.35; 8.39; 8.39; 2.64; 3.08; 2.99; 2.88; 3.02 8.25 9.75 N.R.;
