- Application -
High-Throughput pKa Determination Using the cePRO 9600™ System Jeremy Kenseth, Ph.D. Andrea Bastin Introduction A majority of drugs contain at least one ionizable moiety. Consequently, experimental knowledge of the acid dissociation constant (pKa value) of a drug candidate compound is of vital importance when attempting to understand issues related to ADMET (Absorption, Distribution, Metabolism, Excretion, Toxicity), formulation development and chromatographic and electrophoretic separation behavior. A recent trend has emerged whereby various physicochemical properties including pKa values are determined early in the discovery process in parallel with activity screening.1 This approach can lead to a higher overall success in identifying viable candidates that possess not only high activity but also favorable properties for formulation and delivery. Capillary electrophoresis (CE) is rapidly gaining acceptance in the pharmaceutical industry as an alternative or complementary method to traditional potentiometric or spectroscopic approaches for determining pKa values.2-6 The adoption of CE is attributed to many advantages for pKa screening. For example, no knowledge of sample concentration is required for analysis, and no spectral differences need to exist between the neutral and ionized forms of a compound. Importantly, the minimal sample requirements and less stringency with regards to compound purity when using CE are particularly attractive for early discovery screening, as often only mg
quantities of relatively impure compounds are available. The use of multiplexed, absorbance-based capillary electrophoresis in a 96-capillary format can provide a substantial increase in throughput over single capillary CE methods for screening pKa values. This application note will describe the use of the cePRO 9600™ in a vacuum-assisted CE mode to rapidly determine the apparent pKa’ values of compounds. A set of eight test compounds with varying pKa’ values was selected to demonstrate the method. An evaluation of the run-to-run reproducibility and throughput obtainable with the cePRO 9600™ system will be presented. The software used to extract the electrophoretic data and calculate the resulting pKa’ values will also be described. Experimental All chemicals were purchased from Sigma or Fisher. An eight-component library containing monoacidic, monobasic, and dibasic compounds possessing molecular weights from 94-324 and literature pKa values from 2.8-10.1 was selected for the method demonstration. All sample solutions contained 0.1% dimethyl sulfoxide (DMSO, an EOF neutral marker) and were prepared at 100 ppm (0.1 mg/ml) concentration. Basic compounds were dissolved in 1 mM H3PO4 and acidic compounds were dissolved in 1 mM NaOH. A 12-component buffer series bracketing a pH range from 2.1-10.8 (I = 50 mM) was used for collecting the electrophoretic data.
A 96-capillary array of 33 cm effective length and 55 cm total length (75 µm i.d., 150 µm o.d.) was used for the pKa analysis. The capillary array was initially flushed for 3 min with water followed by 3 min with 10 mM sodium tetraborate (pH 9.1) at 40 psi. The inlet buffer tray contained a matrix of the 12 different pH buffers in replicates of 8 (Figure 1A). Prior to a run, the capillaries were filled with the inlet buffers for 2 min at –2 psi. The sample tray contained 8 different compounds in replicate rows of 12 (Figure 1B). Samples were injected at –0.2 psi for 5 sec. The CE run was performed at 3.5 kV with –0.2 to –0.4 psi vacuum assistance, providing run times of 9 – 13 min. In between runs, the capillary array was pressurized for 1 min at 40 psi with 10 mM sodium tetraborate outlet buffer. The 1 2 3
5
6
7 8
A custom designed software program (pKa Estimator©) was utilized to determine compound apparent (I = 50 mM) pKa’ values. The 96-capillary electrophoretic data was extracted and each set of 12 electropherograms was displayed with labeled analyte and DMSO peaks for visual confirmation. The effective mobility (meff) was determined at each pH value from the migration time difference between the analyte and DMSO. A non-linear regression analysis was then performed on the accepted mobility data, estimating the analytes’ pKa’ value(s).
9 10 11 12
1 2 3
B
4
5
6
7 8
9 10 11 12
4-Aminopyridine
B
C
C
Quinine Salicylic Acid
D
D
Lidocaine
E
E
F
F
G
G
Metoprolol Betahistine Warfarin -
H
H
Aspirin
pH
9.20 10.02 10.86
A
B
7.62 8.43
A
2.14 2.90 3.41 4.41 5.21 6.03 6.82
A
4
resulting cycle time per 8 compound sample plate was approximately 20 min (24 compounds/h).
Figure 1. A: Inlet buffer arrangement in a 96-well tray for 8 compound parallel pKa analysis (deep well plate; 1.2 ml/well). B: Arrangement of 8 compounds in a 96-well sample tray (PCR plate, 100 µl/well). The highlighted well corresponds to metoprolol at pH 7.62. Results The determination of pKa values using CE involves measuring the ionic mobility of a compound as a function of pH. The resulting plot of meff vs. pH is a sigmoidally-shaped titration curve where the inflection point(s) reflect the pKa’ value(s). Using this approach, the overall charge of a compound as a function of pH from 2-11 can be visualized. The multiplexed design of the cePRO 9600™ instrument provides the capability to simultaneously evaluate different pH buffers in different capillaries in a single CE run. As a result, parallel pKa screening of eight different compounds over
12 pH values can be achieved. To increase the pH resolution and expand the pH range, four different compounds can be evaluated simultaneously using a 24-point buffer series from pH 1.8–11.2. Figure 2 shows 96-capillary electrophoretic data collected using the experimental design of Fig. 1. Each 12-capillary row contains a different compound, screened over 12 different pH values. Acidic compounds required approximately 12 min for analysis at a vacuum level of –0.2 psi, while migration data for the basic compounds was obtained in 10 min. The analysis time can be reduced to approximately 8 min for acidic
compounds when using a –0.4 psi vacuum level. However, the resolution of the analyte and marker peaks at pH values near charge neutrality decreases at the higher vacuum
level. Therefore, to achieve the most accurate results the lower –0.2 psi vacuum level is preferred.
Quinine
4-Aminopyridine
Lidocaine
Salicylic Acid
Betahistine
Metoprolol
Warfarin
Aspirin
Figure 2. 8-Compound, 96-capillary electropherogram data obtained using the experimental arrangement described in Fig. 1. For each compound, the set of 12 different electropherograms represents a parallel screen from low pH (upper left) to high pH (lower right). The time scale is set to 13 min. Figure 3 shows the electrophoretic data obtained for acetylsalicylic acid (Row H, capillaries #85-96). Although a substantial salicylic acid degradant impurity is present, the pKa’ value for acetylsalicylic acid can still be obtained because the impurity is readily separated from the target compound. In fact,
the pKa’ value for the salicylic acid impurity can also be determined from the same CE run. This example demonstrates the substantial advantage of CE-based screening methods for the analysis of impure samples.
Figure 3. Parallel pKa screening of acetylsalicylic acid. Electropherograms covering the pH range are displayed from low pH (#85, top left) to high pH (#96, bottom right). The observed migration order is DMSO, acetylsalicylic acid, and salicylic acid (degradant). Figure 4 shows a screen capture from the pKa Estimator© program displaying the 12 extracted electropherograms for 4aminopyridine from pH 2.14 to pH 10.86 (capillaries #1-12). The software automatically selects both the analyte and DMSO peaks. The user is allowed to accept the peak assignments or reverse the order if the compound is negatively charged
(migrates after DMSO). The software applies the best fitting equation to the accepted migration data based on compound molecular weight and limiting mobility,5 and then displays the resulting pKa’ value(s). Figure 5 shows representative meff vs. pH plots for monobasic (4aminopyridine), dibasic (quinine), and monoacidic (warfarin) compounds.
Figure 4. Electropherograms for 4-aminopyridine from pH 2.14 (top left) to pH 10.86 (bottom right) as extracted by the pKa Estimator© software. To accept the current peak assignments, the user selects Accept All 12 and the mobility data is entered for nonlinear regression analysis of the pKa’ value.
4-Aminopyridine (monobasic)
Quinine (dibasic)
Warfarin (monoacidic)
Figure 5. Representative plots of meff vs. pH calculated using the pKa Estimator© software. Table 1 summarizes pKa screening results for the eight different compounds and provides a comparison to literature values. Excellent agreement is obtained when one considers the variations in experimental methods employed. To date, the pKa’ values of over 100 diverse compounds have been
determined using the cePRO 9600™. In general, agreement to within 0.2 pKa units was obtained between cePRO 9600™ determined pKa’ values and available literature values. Results from these studies will be forthcoming.
Literature pKa Values Compound
cePRO 9600™ pKa' Value (I = 50 mM)
SD
4-Aminopyridine
9.17
0.01
9.18
9.02
4.28
0.07
4.18
4.13
4.14
4.22
8.39
0.05
8.35
8.39
8.39
8.55
2.88
3.02
Quinine
Salicylic Acid
2.81
0.02
Lidocaine
7.90
0.04
Metoprolol
9.54
0.02
Betahistine
5
CE (I = 50 mM) CE (I = 0 mM)3 CE (I = 0 mM)4 CE (I = 0 mM)6 UV (I = 150 mM)7 Other
3.08
2.64 7.92
9.51
7.83 9.58
3.99
0.05
5.21
3.46
9.88
0.02
10.13
9.78
Warfarin
4.99
0.02
Acetylsalicylic Acid
3.46
0.02
5.15
4.98 3.47
5.06
4.9 3.3
Table 1. Comparison of cePRO 9600™ and literature pKa values for the eight compound test library (n = 8 runs). An evaluation of the run-to-run reproducibility of the cePRO 9600™ for pKa determination is summarized in Figure 6. Plots of meff vs. pH for 4-aminopyridine
collected over the eight different runs are overlaid, demonstrating the excellent reproducibility obtainable with the method.
8
Effective Mobility (x 106 cm2/V•s)
pKa’ Value
400 350
9.17
250
Run 1 Run 2 Run 3 Run 4
200
Run 5
9.19
150
Run 6 Run 7
9.17
Run 8
9.18
300
100
9.18 9.17 9.14
9.18
50 0 1.5
2.5
3.5
4.5
5.5
6.5
7.5
8.5
9.5 10.5 11.5
9.17 ± 0.01
pH Value Figure 6. Run-to-run reproducibility of effective mobility vs. pH for 4-aminopyridine.
Summary This application note has demonstrated that multiplexed, absorbance-based CE with the cePRO 9600™ system in a vacuumassisted mode can provide a high throughput, accurate, and reproducible method for the pKa determination of acidic and basic compounds. The current throughput is 24 compounds/h (12 pH values) with a pKa screening range of approximately 2-11. If higher pH resolution is desired, a 24-component buffer series from pH 1.8–11.2 can be applied to four compounds simultaneously at a throughput of 12 compounds/h.
(3)
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(5)
(6)
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References (1) (2)
Kerns, E. H. J. Pharm. Sci. 2001, 90, 1838-1858. Caliaro, G. A.; Herbots, C. A. J. Pharm. Biomed. Anal. 2001, 26, 427-434.
Jia, Z.; Ramstad, T.; Zhong, M. Electrophoresis 2001, 22, 11121118. Ishihama, Y.; Nakamura, M.; Miwa, T.; Kajima, T.; Asakawa, N. J. Pharm. Sci. 2002, 91, 933-942. Miller, J. M.; Blackburn, A. C.; Shi, Y.; Melzak, A. J.; Ando, H. Y. Electrophoresis 2002, 23, 28332841. Wan, H.; Holmen, A.; Nagard, M.; Lindbery, W. J. Chromatogr., A 2002, 979, 369-377. Box, K.; Bevan, C.; Comer, J.; Hill, A.; Allen, R.; Reynolds, D. Anal. Chem. 2003, 75, 883-892. Reichard, R. E.; Fernelius, W. C. J. Phys. Chem. 1961, 65, 380-381.
Andrea Bastin is a chemical technician and Jeremy Kenseth is an application scientist and laboratory manager at CombiSep, Inc., Ames, IA.
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Copyright © 2004 CombiSep, Inc. All Rights Reserved. Reproduction, adaptation or translation without prior written permission is prohibited, except as allowed under the copyright laws. Published in original form July 22, 2002 Updated February 13, 2004.
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