Measurement of Dissociation Constants (pKa Values) of
Organic Compounds by Multiplexed Capillary
Electrophoresis Using Aqueous and Cosolvent Buffers
MARINA SHALAEVA,1 JEREMY KENSETH,2 FRANCO LOMBARDO,1 ANDREA BASTIN2
1
Pfizer Global Research and Development, Groton Laboratories, Groton, Connecticut 06340
2
CombiSep, 2711 South Loop Drive, Suite 4200, Ames, Iowa 50010
Received 7 June 2007; revised 15 October 2007; accepted 15 November 2007
Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/jps.21287
ABSTRACT: Evaluation of a multiplexed capillary electrophoresis (CE) method for pKa
measurements of organic compounds, including low solubility compounds, is presented.
The method is validated on a set of 105 diverse compounds, mostly drugs, and results are
compared to literature values obtained from multiple references. Two versions of the
instrument in two different labs were used to collect data over a period of 3 years
and inter-laboratory and inter-instrument variations are discussed. Twenty-four
point aqueous and mixed cosolvent buffer systems were employed to improve the
accuracy of pKa measurements. It has been demonstrated that the method allows
direct pKa measurements in aqueous buffers for many compounds of low solubility,
often unattainable by other methods. The pKa measurements of compounds with
extremely low solubility using multiplexed CE with methanol/water cosolvent buffers
are presented. ß 2008 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci
Keywords: pKa measurements; multiplexed capillary electrophoresis; low solubility
compounds; mixed cosolvent buffers; aqueous buffers; dissociation constants of drug
molecules
INTRODUCTION
The acid–base dissociation constant of substances
(pKa value) is a very important parameter in drug
design and optimization. The degree of ionization
strongly affects solubility, permeability, and drug
This article contains supplementary material, available at
www.interscience.wiley.com/jpages/0022-3549/suppmat.
Advanced Analytical Technologies, Inc. (Formerly CombiSep), 2711 South Loop Drive, Suite 4200, Ames, IA 50010.
Franco Lombardo’s present address is Novartis Institutes for
Biomedical Research, 250 Massachusetts Avenue, Cambridge,
MA 02139.
Correspondence to: Franco Lombardo (Telephone: 617-8714003; Fax: 617-871-3078.; E-mail: franco.lombardo@novartis.
com)
Journal of Pharmaceutical Sciences
ß 2008 Wiley-Liss, Inc. and the American Pharmacists Association
disposition properties—absorption, distribution,
metabolism and excretion (ADME).1 For example,
our recent work on the prediction of volume of
distribution in humans (VDss)2 has shown that
the fraction of compound ionized at pH ¼ 7.4,
together with the fraction of free drug in plasma
are the largest contributors to the prediction of
VDss, via the fraction unbound in tissues ( fut).
In recent years high throughput drug discovery
operations have brought to focus the need for
the rapid evaluation of various physicochemical
properties of newly synthesized compounds using
minute quantities3–6 and significant efforts have
been devoted to developing techniques adapted
to these challenges. There have been a number of
methods employed for pKa measurements based
on solubility, potentiometric titration,7–10 spectrophotometry,11–14 HPLC15–17 and, most recently,
JOURNAL OF PHARMACEUTICAL SCIENCES
1
2
SHALAEVA ET AL.
on capillary electrophoresis (CE).18–33 The solubility method is of limited accuracy and potentiometric titrations typically require mg amounts of
pure sample with a throughput of 20–40 min per
compound.34 In addition, the sample concentrations required for potentiometry often result in
precipitation of poorly soluble compounds, necessitating mixed solvent extrapolation methods and
further impacting sample throughput.
A rapid method for pKa measurement employing
a mixed-buffer linear pH gradient and spectrophotometric detection was recently demonstrated.35 The spectral gradient analysis (SGA)
technique performs pKa measurements sequentially from a 96-well plate format using 10 mM
DMSO stock solutions and samples are assayed in
about 4 min. Compounds, however, must possess
good chromophores close enough to the center of
ionization for the detection of a spectral shift with
changing pH; otherwise, the pKa values may go
undetected. Precipitation of low solubility compounds is also a limitation of the method, although
cosolvents could be employed, but data analysis
often requires user intervention and sample
impurities, degradants or counter ions possessing
similar UV characteristics may also potentially
interfere with the measurement.
The application of CE for medium to high
throughput pKa measurements in support of
drug discovery projects has steadily increased
in recent years. The many advantages of CE
for pKa measurement were highlighted in several
recent reviews34,36–38 including: minimal sample
requirement with extremely low sample consumption, the ability to separate impurities and/or
degradants from the target compound, sensitive
on-line UV detection, and automated operation.
In addition, precise knowledge of sample concentration is not required because only compound
migration times are required for analysis, and no
special demands are placed upon the purity of
buffer solutions.
The effective mobility (meff) of a solute, in a
field of voltage V, is easily calculated from the
difference in migration time between the compound (ta) and a neutral marker (tm), commonly
DMSO, through Eq. (1):
Ld Lt
1
1
meff ¼
(1)
ta tm
V
and this technique is based on the variation of the
solute mobility with the variation of buffer pH.
In Eq. (1) Ld is the length from the capillary
inlet to the detection window and Lt is the total
JOURNAL OF PHARMACEUTICAL SCIENCES
capillary length in cm, and V is the applied
voltage. A plot of compound meff as a function of pH
yields a sigmoidal curve, from which the inflection
point(s) corresponds to the apparent pKa value(s)
and thus the mobility is correlated with the pKa.
Equations relating the measured meff and pH
values to the pKa value via nonlinear regression
analysis have been previously well described in
the literature for up to three ionizable groups,
which covers the majority of pharmaceutical
compounds.22,28 Theoretically, there is no restriction on the number of ionization equilibria that
can be considered.34
In Pharmaceutical Discovery operations it is
often of interest to discriminate differences in pKa
values among structural analogs and it is not
unusual to encounter molecules with two or more
very close pKas. For the method to be suitable for
the simultaneous measurement of the variety of
possible pKa combinations, in a high throughput
mode, it is necessary to have buffers spaced with
relatively small increments and also covering as
wide a pH range as possible. For example,
Ishihama et al.22 demonstrated the successful
measurement of up to six pKa values for angiotensin by CE using 19 different buffers from pH
1.8 to 12.0.
One approach to increasing sample throughput
involves the application of multiplexed capillary
array electrophoresis with UV absorption detection.39,40 Recently, the use of a 96-capillary
array for the rapid measurement of pKa values
for 96 different compounds (mostly monoprotic
acids and bases) was demonstrated.33 A measurement of 128–168 compounds in an 8 h period was
achieved by simultaneously analyzing eight compounds over 12 pH values in a single CE run.
A significant challenge for all of the aforementioned pKa measurement techniques including CE is the precipitation of low aqueous
solubility compounds. Unfortunately, the current
trend in drug discovery is toward compounds
possessing a relatively high lipophilicity and a
fairly low aqueous solubility.41 Detection limits
using UV spectrophotometry are reported to be
1–10 mM as long as the compound possesses a
suitable pH-sensitive chromophore,5 while CE
methods employing low UV wavelength detection
allow measurements at similar concentrations
without restrictions regarding the position of the
chromophore.22
A potential approach for increasing the detection sensitivity and expanding the scope of
detection involves the integration of mass spectroDOI 10.1002/jps
MULTIPLEXED CE pKa DETERMINATION
metry with CE (CE-MS).42,43 Wan et al.43 measured the pKa values for 60 different compounds
including some that were sparingly soluble or of
low UV absorbance by CE-MS, and sample
throughput was increased by pooling up to 56
compounds. Although promising, the day-to-day
reliability and reproducibility of the method
remains to be assessed and commercially available software for performing mass deconvolution
and data processing would need to be developed to
enable routine pKa measurement.
A common approach for the pKa measurement
of aqueous insoluble compounds involves the use
of different percentages of mixed cosolvent buffers
and extrapolation to 0% cosolvent. Typically, at
least three and up to six different percentages of
cosolvent are recommended. Methanol is the most
commonly employed cosolvent and is considered
to yield the least deviation from a completely
aqueous environment as its effects on pKa have
been extensively studied.9,44,45
When working in mixed methanol/water solutions two different pH scales are commonly used,
depending upon how the pH meter is calibrated
prior to pH measurement. The notation describing
the different pH scales used within this work is
that recommended by IUPAC for pH quantities46
and that employed by others.45,47–49 The lowercase left-hand superscript indicates the solvent
(w for water or s for mixed solvent) in which the
measurement is made, while the lower-case lefthand subscript indicates the solvent where the
ionic activity coefficient is referred to unity at
infinite dilution (i.e., how the meter was calibrated). Therefore, the measurement of pH values
following calibration of the pH meter with
standard buffers prepared, in the same methanol/water composition as the sample, yields ss pH
values. If the pH meter is calibrated with aqueous
standard buffers, measurements in the mixed
methanol/water solutions are in the intersolvental
scale and referred to as sw pH values. It has been
rigorously demonstrated in the literature that the
two pH scales are related by means of Eq. (2):
s
s pH
¼ sw pH þ d
(2)
where d is a constant that is dependent strictly
upon the composition of the mixed solvent.47
Eq. (2) assumes that the liquid junction potential
of the potentiometric system used for pH measurement is negligible, as was previously demonstrated for the same type of combination glass
electrode used in this work.47 Values for d have
DOI 10.1002/jps
3
been previously determined over a wide range of
methanol/water compositions.48
Measurements of meff as a function of sw pH value
using CE yield a compound’s apparent sw pKa
value(s) for that particular solvent composition
and ionic strength. The sw pKa value can be
converted to a ss pKa value via Eq. (3):
s
s pKa
¼ sw pKa þ d
(3)
Alternatively, the ss pKa value can be obtained
directly for a compound working in the ss pH scale
by employing CE with cosolvent buffers prepared from equimolar mixtures of an acid and its
corresponding salt50–52 or by using pH meter
calibration standards with known ss pKa values
(e.g., potassium hydrogenphthalate).53,54 However, use of the sw pH scale combined with
Eqs. (2) and (3) provides a much more practical
approach when working with mixed solvent
systems.
The most popular extrapolation method is the
Yasuda–Shedlovsky (Y–S) method, which relates
s
s pKa to the inverse of the dielectric constant of the
binary solvent (1/e) by Eq. (4):
s
s pKa
þ log½H2 O ¼
a
þb
"
(4)
where a and b are constants. Extrapolation to
e ¼ 78.3 and log H2O ¼ 55.5 (the dielectric constant
and molar concentration of pure water, respectively) yields the apparent w
w pKa value. Previous
studies have demonstrated that Y–S extrapolation generally yields a linear relationship and
accurate w
w pKa values when using solvent mixtures
possessing e > 50, which for methanol corresponds
to <60 wt% or <65.5% (v/v).5,44
Although numerous publications have appeared describing the application of CE for performing
aqueous pKa measurements, relatively few have
explored the use of methanol/water cosolvents.
Bellini et al.55 described the measurement of pKa
values in increasing methanol concentrations and
extrapolation to 0% cosolvent on a set of five acids.
Due to the limited throughput (up to 40 min per
separation) the ‘‘two pH points per compound at
four different methanol concentrations’’ approach
was recommended as more practical, requiring
only eight runs per compound. Buckenmaier
et al.45 measured by CE the sw pKa values of
several basic compounds commonly used as test
probes in HPLC over varying methanol contents.
A few other studies have measured the ss pKa values
for compounds using CE in pure methanol.50–52
JOURNAL OF PHARMACEUTICAL SCIENCES
4
SHALAEVA ET AL.
In the present work we report and discuss
the measurement of compound pKa values with
multiplexed 96-capillary array electrophoresis
employing fixed wavelength UV detection at
214 nm and 24 pH point aqueous or methanol
cosolvent buffers. The goals of this study were
to validate the method employing a diverse set
of acidic, basic, and multiprotic compounds; to
evaluate issues related to the number of pH
buffers used and its relation to the accuracy and
the resolution of closely spaced pKa values;
to assess the compound solubility limits for
aqueous pKa measurement; and to investigate
the use of cosolvent extrapolation methods for
the measurement of insoluble compounds by
multiplexed CE. As a test set we used almost
exclusively drug compounds spanning a wide
range of structures, physico-chemical properties
and, importantly, containing many examples of
multiprotic compounds.
EXPERIMENTAL
Apparatus
Two different multiplexed 96-capillary array
electrophoresis systems (Advanced Analytical
Technologies, Inc., Ames, IA) were utilized in this
study: a MCE 2000 (Pfizer Laboratory, Groton,
CT) and a cePRO 9600TM (Advanced Analytical
Laboratory), the first and second generation
systems, respectively, employing slightly different configurations and experimental methods as
discussed in the procedures below. Only a brief
description of the instrument is given here; a more
complete discussion can be found in earlier
reports.33,39,40
pH measurements were taken with a Ross
8102 combination glass electrode, equipped with
an automatic temperature compensation (ATC)
probe, using a Thermo Orion 520Aplus pH meter.
The relative pH accuracy of the meter was specified to be 0.002 pH units.
Chemicals
The test set compounds were ordered from various
commercial sources including Sigma–Aldrich
(St. Louis, MO), ICN (Irvine, CA), and Sequoia
(Pangbourne, UK), and were used as received.
HPLC grade water from Baker (Phillipsburg, NJ)
was used throughout. All other common reagents
were purchased from Sigma–Aldrich and used as
received.
JOURNAL OF PHARMACEUTICAL SCIENCES
Aqueous and methanol/water mixed cosolvent
pH buffer kits along with a common 10 outlet
reservoir buffer concentrate (100 mM sodium
tetraborate) were prepared by Advanced Analytical. The 10 outlet buffer concentrate was
diluted with doubly distilled H2O prior to use.
The sets of 24 aqueous pH buffers were prepared
from phosphoric acid, formic acid, sodium acetate,
sodium phosphate, or boric acid with the addition
of sodium chloride or/and sodium hydroxide in
various proportions to a level ionic strength of
I ¼ 50 mM. The sets of mixed cosolvent buffers
(also I ¼ 50 mM) were prepared from similar
inorganic buffers with the addition of 30%, 40%,
50%, or 60% (v/v) methanol. The pH values of the
mixed cosolvent buffers were measured after
mixing with methanol using a pH meter calibrated with standard aqueous buffers. The
measured values are therefore described as sw pH
values (see Introduction Section).17,47,48 The sets
of aqueous and mixed cosolvent buffers were
stored in the lab tightly closed in the plastic
bottles and used for a period of 3–5 months in most
cases. The pH values of the buffers were generally
stable to 0.04 pH units when stored tightly
sealed between uses. To ensure the highest level of
accuracy over long term storage, particularly in
the buffers above pH 9.0, it is recommended to
periodically measure the pH values using a
calibrated pH meter.
Sample Preparation
Samples were prepared by weighing 0.2–1.0 mg of
compound followed by addition of solvent to yield a
concentration of 50–150 mg/mL (ppm). In the case
of some low solubility compounds, samples could
be further diluted to approximately 5–10 mg/mL to
avoid precipitation in aqueous buffer experiments. The solvent consisted of 0.1–0.2% (v/v)
DMSO (neutral marker) and 1–5 mM HCl or
NaOH to enhance the solubility of basic or acidic
compounds, respectively. Some low solubility
compounds were initially solubilized in 60% (v/v)
methanol prior to addition of aqueous solution.
A few compounds (e.g., indomethacin) were
observed to appreciably degrade following preparation in HCl or NaOH containing solvents (see
Results and Discussion Section). Sample solutions
were sonicated for 30 s and filtered through a
0.20 mm nylon syringe filter if necessary. When
performing mixed cosolvent experiments it was
beneficial to prepare samples at the lowest
DOI 10.1002/jps
MULTIPLEXED CE pKa DETERMINATION
possible concentration and in the presence of 60%
methanol (v/v) to minimize effects from compound
precipitation and improve the peak shape of the
DMSO marker peak during the separation.
Procedures
Prepared sample solutions were dispensed into a
300 mL Costar 96-well microplate (300 mL/well) or
into a 200 mL skirted PCR plate (50 mL/well)
depending on the instrument used. A 24 point pH
experiment required 24 wells to be filled with each
sample and, with this protocol, four compounds
could be analyzed simultaneously.
A deep well (1.0 mL working volume) 96-well
microplate was employed as the pH buffer plate
for analysis. The buffer plate was prepared fresh
for each experiment. Each of the 24 pH buffers
(1.1 mL/well) was dispensed consecutively in wells
A1–B12 for the first sample, wells C1–D12 for the
second sample and so forth.
The buffer plates utilized in the mixed cosolvent
experiments were prepared in a similar layout to
the aqueous buffer plates. Each cosolvent buffer
plate was prepared with a common percentage
of methanol (v/v): 60%, 50%, 40%, or 30%. The
sample plate was sequentially analyzed with each
cosolvent buffer plate, from 60% to 30% methanol
content, to acquire the corresponding sw pKa
values.
pKa Measurements
Experiments performed with the MCE 2000
system used a 96-capillary array of bare fused
silica capillaries (75 mm i.d., 150 mm o.d.) with
effective and total lengths of 55 and 80 cm,
respectively. Prior to each experiment the capillaries were flushed with water followed by the
sodium tetraborate outlet buffer (pH ¼ 9.0) for
5 min each at 40 psi. Following the capillary flush,
the pH buffer plate was loaded and the capillaries
were filled with the different pH buffers for 8 min
at 2.0 psi vacuum. The sample plate was next
inserted into the instrument and the samples
were injected at 1.0 psi for 5 s. The buffer plate
was placed back into the instrument immediately
after sample injection and the electrophoresis
separation started. Aqueous pKa measurements
were performed at þ6 kV for 18 min, while
cosolvent measurements were run at þ5 kV for
25 min, with 0.5 psi vacuum level applied during
CE in both cases.
DOI 10.1002/jps
5
Experiments performed on the cePRO 9600TM
system utilized slightly different operating conditions. The 96-capillary array of bare fused silica
capillaries (75 mm i.d., 150 mm o.d.) had effective
and total lengths of 33 and 55 cm, respectively.
The capillary array was flushed with water
followed by the sodium tetraborate outlet buffer
(pH ¼ 9.0) each morning for 3 min at 50 psi. At the
start of an experiment the pH inlet buffer plate, an
empty 1.0 mL 96-well waste plate and up to two
96-well sample plates are loaded onto the stage.
The capillaries are initially flushed with outlet
buffer for 1 min at 50 psi and then the capillaries
are filled with the different pH buffers for 2 min
at 2.0 psi vacuum and, subsequently, samples
are injected at 0.1 psi for 20 s. The inlet pH
buffer plate is returned into position and the
separation is started. For aqueous experiments
the separation was performed at þ3.5 kV for
13 min with a 0.2 psi vacuum applied during CE.
For cosolvent experiments, the samples were
injected at 0.1 psi for 50 s and separated at
þ3.5 kV for 18 min with the addition of 0.25 psi
vacuum. No flushing of the capillaries was
required between runs with the exception of the
1 min outlet buffer preflush at the beginning of
each analysis cycle.
pKa Data Analysis
Versions 1.01–4.03 of the program pKa EstimatorTM (Advanced Analytical Technologies, Inc.)
were used to calculate the pKa values of compounds from the electrophoresis data. The separation data were imported into the program where
the effective mobility (meff) was calculated for each
pH value from the difference in migration times of
the compound (ta) and DMSO marker (tm) using
Eq. 1. The meff was then plotted as a function of pH
to yield a sigmoidally-shaped titration curve. The
molecular weight (MW) of the compound was
entered into the software and the compound
charge (Zc) was estimated by the software using
a previously described empirical relationship
between MW, maximum meff and Zc.28 From the
calculated compound charge, a suitable equation
from a set of nine different model equations
representing up to three ionizable groups28,22 was
selected by the software to perform a nonlinear
regression analysis on the data. When performing
a cosolvent experiment, the final result is the
apparent sw pKa (I ¼ 50 mM) at the particular %
methanol concentration. Each sw pKa result is
JOURNAL OF PHARMACEUTICAL SCIENCES
6
SHALAEVA ET AL.
converted to its respective ss pKa value by use of the
appropriate d term.48 The apparent ss pKa values
are then plotted as a function of the reciprocal of
the solvent dielectric constant (1/e) (Y–S method)
9,48,56,57
to 0% cosolvent to yield the apparent w
w pKa
value (I ¼ 50 mM) according to Eq. (4).
RESULTS AND DISCUSSION
1. Aqueous pKa Measurements
Comparison to Literature Values
The test set compiled for the multiplexed CE
method validation included a total of 105 compounds (166 total pKa values) of which most were
commercially available drugs. Table 1 lists the
results for compounds that were successfully
measured with aqueous buffers (98 of 105). An
additional seven compounds were found to precipitate during aqueous analysis and their pKa
values were measured using methanol cosolvent
buffers (see Experimental Section). We attempted
to make the validation set as diverse as possible to
thoroughly evaluate various combinations of
physicochemical properties encountered within
a Pharmaceutical Discovery setting. Nearly half
(49 of 105) of the compounds in the validation set
are multiprotic, having from two to five pKa
values. Included are basic, acidic, and zwitterionic
compounds.
The range of pKa values measurable by multiplexed CE employing the 24-point aqueous buffers
was approximately equal to the pH range of the
buffer series (pH 1.75–11.20). Accordingly, we
were able to measure several pKa values below 2.0
(buspirone pKa ¼ 1.93; nicotinic acid pKa ¼ 1.86;
piroxicam pKa ¼ 1.81; sulfacetamide pKa ¼ 1.26)
and above 11.0 (cefuroxime pKa ¼ 11.30; sulfasalazine pKa ¼ 11.31). We were unable to reproducibly fit the lower pKa value for leucovorin
(previously not reported) and the uppermost pKa
value for terbutaline (literature pKa 10.58–11.10;
see Tab. 1). These results are consistent with
previous studies which found that it was possible
to fit pKa values from data encompassing only a
portion of the titration curve.20,22
All of the compounds in the test set were
well detected at concentrations of 50–100 ppm
(mg/mL). However, several compounds (identified
by superscript a) were observed to precipitate at
the typical working concentration when the
pH value approached that corresponding to the
neutral form of the compound. The sensitivity
JOURNAL OF PHARMACEUTICAL SCIENCES
provided by low UV detection at 214 nm allowed
for dilution of these insoluble compounds up to
10-fold and for successful measurement of their
pKa values in aqueous buffers without precipitation. Methanol cosolvent buffers were employed to
analyze compounds that were still observed to
precipitate at concentrations approaching their
detection limits.
Compounds were typically dissolved in 1 mM
HCl (for bases) or 1 mM NaOH (for acids) to
improve their solubility during sample preparation. However, a few compounds were observed to
partially degrade during the time course of the
analysis when dissolved in 1 mM acid or base
(identified by superscript b). As it could be assumed
that the intact compound would possess the
highest MW relative to any degradation products
and therefore the lowest meff it was possible in most
cases to measure the pKa value of the intact
compound in the presence of degradant impurities.
Alternatively, preparation of unstable compounds
in water was also found to minimize degradation.
For the majority of compounds in the test set
multiple literature pKa values have been reported
using a variety of experimental methods and
conditions. We felt it was important and most
appropriate to include all verifiable results in our
comparisons as opposed to citing results from a
single source or method. This approach allows
for a more complete assessment of our results in
comparison to previous studies.
The pKa values measured in this work are
reported as apparent pKa values at I ¼ 50 mM. An
important factor to note when comparing experimental data is that the pKa literature values are
reported over a typical range of ionic strengths
from I ¼ 0 mM (thermodynamic) to 150 mM. When
converting from an ionic strength of 150 to 0 mM
the correction factor at 258C would be þ0.12 for a
monoacid and 0.12 for a monobase. A majority
of potentiometric and spectroscopic data are
reported as apparent pKa values at I ¼ 150 mM.
The correction between pKa values at I ¼ 50 mM
measured in this work and I ¼ 150 mM would be
0.04 for monoacids or monobases, 0.11 for the
weaker pKa (z ¼ 2) of diacids or dibases, and 0.18
for the weakest pKa (z ¼ 3) of triacids or tribases. A
significant amount of literature results have been
converted to I ¼ 0 mM, especially for CE-based
measurements. For the weaker pKa of a diacid or
dibase, conversion from I ¼ 150 mM to I ¼ 0 mM
represents a span of 0.36 pKa units. Some
references either do not mention the ionic
strength or do not clearly state whether or not a
DOI 10.1002/jps
MULTIPLEXED CE pKa DETERMINATION
7
Table 1. Summary of pKa Values Measured by 96-Capillary Array CE Using Aqueous Buffers and a Comparison to
Literature Values
Compound
1
2
3
4
5
Abacavir
Acebutolol
Acetaminophen
(paracetamol)
Acetylsalicylic acid
Acyclovir
6
7
Alprenolol
4-Aminobenzoic acid
8
9
10
11
12
Aminopyridine, 2Aminopyridine, 4Amitriptylinea
Atenolol
Betahistine
13 Bifonazole
14 Buspirone
15 Cefadroxil
16 Cefuroximeb
17 Cephalexinb
18 Cetirizine
19 Chloroamphetamine
20 Chloroquine
21 Chlorthalidone
22
23
24
25
Cimetidine
Clomipraminea
Clotrimazolea
Clozapine
26
27
28
29
Codeine
Deprenyl
Desipramine
Dichlorphenamide
30 Diphenhydramine
31 Emetine
32 Eserine
(physostigmine)
33 Flufenamic acida
34 Flumequine
DOI 10.1002/jps
pKa Average, pKa Average,
This Work
Literature Difference pKa Range
References
5.04
9.52
9.41
5.01
9.48
9.56
0.03
0.04
0.15
5.01
9.37–9.56
9.45–9.75
66
27,33,35,67,68
14,27,33,35,43,59,69
3.49
2.20
9.18
9.62
2.17
4.76
6.71
9.22
9.51
9.60
3.90
10.02
6.29
1.93
7.64
2.55
7.21
9.71
2.14
11.30
2.61
7.08
2.24
3.47
7.90
9.85
7.44
10.68
8.98
10.82
6.90
9.57
5.99
4.02
7.60
8.15
7.47
10.42
8.24
9.50
9.23
6.37
8.81
8.15
3.50
2.23
9.22
9.52
2.46
4.81
6.72
9.19
9.41
9.54
4.26
9.98
5.8
0.01
0.03
0.04
0.10
0.29
0.05
0.01
0.03
0.10
0.06
0.35
0.04
0.49
7.6
2.66
7.38
9.89
2.11
0.04
0.11
0.17
0.19
0.02
3.30–3.74
27,28,30,35,69
2.16–2.34
5,28,35,70
9.04–9.31
9.38–9.59
27,30,35,68
2.45–2.46
5,12
4.62–4.99
6.70–6.76
21,28,34
9.02–9.29
21,26,28,34
9.32–9.49
5,45,51,71,72
9.42–9.64 26,27,28,29,30,33,35,43,67,69,73,74
3.46–5.21
27,75,76
9.78–10.13
5.72–5.88
77 c
67,69
7.60
2.47–2.86
23,28
7.14–7.59
9.89
2.04–2.17
23
2.66
7.02
2.11
2.91
7.99
9.80
8.25
10.37
9.11
10.98
6.82
9.28
5.89
4.08
7.82
8.11
7.43
10.27
8.3
10
9.13
7.02
8.57
8.15
0.05
0.06
0.13
0.56
0.09
0.05
0.81
0.31
0.13
0.16
0.08
0.29
0.10
0.06
0.22
0.04
0.04
0.15
0.06
0.50
0.10
0.65
0.23
0.00
2.34–3.11
6.79–7.14
2.10–2.12
2.90–2.93
7.98–8.00
9.80
8.10–8.50
9.94–10.87
9.11
10.98
6.68–6.97
9.17–9.38
5.48–6.30
3.58–4.40
7.63–7.94
7.81–8.22
7.40–7.48
10.16–10.65
8.20–8.41
9.82–10.27
9.10–9.16
6.68–7.36
8.23–8.91
8.13–8.17
3.97
6.31
4.02
6.36
0.05
0.05
3.63–4.27
6.09–6.65
23,27,35,59
78,79
80
81,82,83,84
35
27,28,33,43
71 c
85 c
5,43 c
5,29,33,43,45,69
5,68,69
5,35,51,72,74,86
28,87 c
5,45
28,88
14,35
5,27,33,67
5,14,26,28,33,35,43,54,59,69
(Continued)
JOURNAL OF PHARMACEUTICAL SCIENCES
8
SHALAEVA ET AL.
Table 1. (Continued )
Compound
35 Furosemide
36 Histamine
37 Hydroquinine
38
39
40
41
Ibuprofen
Imipraminea
Indomethacinb
Ketoconazole
42 Labetalol
43 Lamivudine
44 Lansoprazole
45 Leucovorin
(folinic acid)
46 Levallorphan
47 Lidocaine
48 Maprotiline
49 Mebendazole
50 Methotrexate
(amethopterin)
51 Metoprolol
52 Metronidazole
53 Morphine
54 Nafronyl
55 Naloxone
56
57
58
59
Naproxen
Nefazodonea
Nefopam
Nicotine
60 Nicotinic acid
61 Nifedipine
62 Norephedrine
63 Norfloxacin
64 Nortriptyline
65 Omeprazole
pKa Average, pKa Average,
This Work
Literature Difference pKa Range
References
3.60
10.35
5.80
9.96
4.32
9.06
4.35
9.58
4.18
3.18
6.30
7.35
9.16
4.24
4.22
8.58
<1.5
3.55
10.46
6.04
0.05
0.11
0.25
3.34–3.74
10.15–10.90
6.04
14,27,28,33,35,43,70,74,89
4.29
9.01
4.34
9.5
4.29
3.15
6.39
7.46
9.38
4.3
3.99
8.79
0.03
0.05
0.01
0.08
0.11
0.03
0.09
0.11
0.22
0.06
0.23
0.21
4.29
9.01
4.01–4.51
9.34–9.66
4.06–4.51
2.90–3.29
6.22–6.51
7.42–7.48
9.32–9.42
4.30
3.82–4.15
8.73–8.84
82
3.59
5.12
10.39
9.26
10.74
7.96
10.57
3.27
9.56
3.30
3.10
4.68
10.28
9.29
0.49
0.44
0.11
0.03
3.10
4.56–4.80
10.15–10.40
8.81–9.77
7.91
10.20
3.43
9.93
3.24
0.05
0.37
0.16
0.37
0.06
7.83–7.98
10.20
3.43
9.93
3.04–3.37
3.9
5.23
9.51
2.38
8.19
9.3
0.04
0.08
0.09
0.11
0.12
0.10
3.80–4.00
4.99–5.39
9.36–9.60
2.38
8.17–8.21
9.26–9.33
7.94
9.44
4.13
0.15
0.41
0.14
7.94
9.44
4.01–4.20
8.98
3.14
8.14
2.18
4.74
2.60
0.64
0.14
0.06
0.33
0.03
0.43
8.65–9.31
3.00–3.25
8.02–8.29
2.00–2.43
4.62–4.84
2.60
6.23
8.49
10.11
4.13
0.09
0.18
0.18
0.12
5.94–6.34
8.22–8.75
10.02–10.19
3.94–4.40
3.86
5.31
9.60
2.49
8.07
9.20
9.06
8.09
9.03
4.27
6.76
8.34
3.28
8.20
1.86
4.71
2.17
9.12
6.14
8.31
10.29
4.25
JOURNAL OF PHARMACEUTICAL SCIENCES
35,90
14,27,28,29,34,35,43,59,70,89,91,92
5,34,35,43,49,51,72 c
5,27,33,35,43,66,93
28,35,43,94
14,35,89,95
96
30,97,98
5,99
60
cd
29,33,35,43,86,91,92
49
43
5,28,43
27,30,33,35,73,74
33
59,69,91
60
27,33,74,89
100
21,26,28,29,34,43
14,29,30,33,35,43,101
28
5,28,35,54,66,102,103,104
5,45,49,51
30,43,97,105,106
DOI 10.1002/jps
MULTIPLEXED CE pKa DETERMINATION
9
Table 1. (Continued )
Compound
66 Pantoprazole
67 Papaverine
68 Penicillin Gb
69 Perfenazine
70 Phenylalanine
71 Pindolol
72 Piroxicam
73 Prazosin
74 Procaineb
75 Promazinea
76 Promethazinea
77 Propranolol
78 Pyrilamine
79 Quinine
80 Salicylic acid
81 Sotalol
82 Sulfacetamide
83 Sulfasalazine
84 Sulpiride
85 Terbutaline
86 Tetracaine
87 Tetracycline
88 Theophylline
89 Thiopropazate
90
91
92
93
Trazodone
Trimethoprim
Trimipraminea
Tripelennamine
94 Tryptophan
DOI 10.1002/jps
pKa Average, pKa Average,
This Work
Literature Difference pKa Range
8.66
3.83
8.04
6.33
2.64
3.64
7.76
2.16
9.01
9.49
1.81
5.28
7.12
2.14
9.05
9.56
9.05
9.59
8.86
3.77
8.19
6.34
2.75
3.59
7.82
2.19
9.08
9.64
2.25
5.18
7.08
2.28
9.06
9.11
8.93
9.51
0.21
0.06
0.15
0.01
0.11
0.04
0.06
0.03
0.07
0.15
0.45
0.10
0.04
0.14
0.01
0.45
0.12
0.08
8.70–8.98
3.56–3.92
8.18–8.19
6.21–6.49
2.75
3.59
7.82
2.18–2.20
9.08
9.54–9.74
1.88–2.53
4.94–5.32
7.04–7.11
2.27–2.29
9.01–9.15
8.92–9.40
8.62–9.10
9.14–9.61
4.14
9.12
4.29
3.99
9.14
4.21
0.15
0.02
0.08
3.99
9.10–9.18
3.95–4.46
8.53
2.85
8.38
9.47
1.26
5.32
2.27
7.87
11.31
9.04
10.10
8.79
9.54
10.47
2.29
8.50
3.36
7.09
9.17
8.51
3.07
7.66
6.80
6.88
9.56
4.08
9.10
2.30
8.51
2.90
8.29
9.82
1.85
5.23
2.53
7.95
11.2
9
10.02
8.58
9.95
10.89
2.3
8.44
3.31
7.51
9.54
8.62
3.2
7.15
6.75
6.84
9.25
4.20
8.71
2.38
0.02
0.05
0.09
0.36
0.59
0.09
0.27
0.08
0.11
0.04
0.08
0.21
0.41
0.43
0.01
0.06
0.05
0.42
0.37
0.11
0.13
0.51
0.05
0.04
0.31
0.12
0.39
0.08
8.35–8.60
2.64–3.08
8.25–8.35
9.72–9.98
1.76–1.95
5.16–5.30
2.40–2.65
7.91–9.70
10.51–11.8
8.95–9.13
9.79–10.19
8.23–8.72
9.89–10.00
10.58–11.10
2.20–2.39
8.29–8.55
3.30–3.33
7.16–7.70
9.43–9.69
8.56–8.66
3.20
7.15
6.69–6.79
6.6–7.07
9.15–9.37
4.20
8.71
2.30–2.60
References
30,97,106
14,26,29,33,35,43,59,67,107
108
109
91,110
33,107
5,27,33,35,74,103
5,35
33,35,67,69,92
72,109
5,68,72,109
27,28,29,30,35,43,67,68,70,74,92,
107
34,67
13,14,21,26,28,29,33,35,43,45,59,
68,82,91
21,26,27,28,30,33,34,35,43,59,69
35,107,111
14,35,43
5,112
27,33,35,66,113,114
13,14,27,35,43,74,107
35,66,92
35,115,116
27,35,91
117
33,49 c
66 c
49 cd
86
22,26,27,28,29,43
(Continued)
JOURNAL OF PHARMACEUTICAL SCIENCES
10
SHALAEVA ET AL.
Table 1. (Continued )
Compound
95 Tyrosine
96 Vancomycin
97 Verapamila
98 Warfarin
pKa Average, pKa Average,
This Work
Literature Difference pKa Range
9.22
2.18
8.76
10.03
2.78
6.96
8.37
8.76
10.06
8.93
4.97
9.35
2.19
9.12
10.21
2.66
7.49
8.63
9.26
10.16
8.81
4.94
0.14
0.01
0.36
0.18
0.12
0.53
0.26
0.50
0.10
0.12
0.03
8.97–9.43
2.18–2.2
8.94–9.21
9.99–10.47
2.66
7.49
8.63
9.26
10.16
8.66–9.07
4.70–5.15
References
22,35,66,91,118
107
5,22,27,28,35,58,74,109
5,22,26,27,28,29,33,35,43,92
c
a
Analyzed at 10–50 ppm to reduce compound precipitation.
Some compound degradation was observed when prepared in 1 mM acid or base.
c
Potentiometric measurement performed by pION, Inc. (Woburn, MA).
d
Potentiometric measurement Pfizer, Sandwich Laboratories, Sandwich (UK).
b
correction was applied. Also, the correction for
zwitterionic compounds is not straightforward
and often authors report apparent pKa values for
zwitterions while correcting acidic and basic
compounds to I ¼ 0 mM.
Another source of data variability stems from
the temperature during the experiment. For our
measurements, room temperature air was circulated around the capillary array by cooling fans in
the instrument during the CE separation. In addition, relatively low field strengths (60–80 V/cm)
were employed to reduce the effects of Joule
heating and maintain a constant temperature of
approximately 20–258C. Indeed, the current level
reached a steady state within 1–2 min and did not
significantly increase throughout the CE separation. In some cases the temperature effect may be
significant, as when Hasegawa et al.58 reported
a shift of 0.33 pKa units for verapamil when
increasing from 20 to 378C. Nevertheless, temperature is often omitted in the literature data
and we used pKa values as found or within the
range of 20–258C if there was a choice.
The medium employed when performing pKa
measurements (i.e., aqueous or cosolvent) may
also slightly influence results relative to others.
Previously reported pKa values for many sparingly soluble compounds listed in Table 1 were
obtained with potentiometry using Y–S cosolvent
extrapolation methods. It has been previously
shown that the Y–S method slightly underestimates pKa values for basic compounds and
overestimates pKa values for acidic compounds
when compared to aqueous-based measurements
(see Sample Preparation Section).59
JOURNAL OF PHARMACEUTICAL SCIENCES
The variability in experimental methods, conditions, and data presentation described above
inevitably brings additional challenges when comparing results. For this reason, we decided to
present not only the average of pKa values found in
the literature, but also present the range of reported
values as seen in Table 1. We found that the average
range in literature values for the compounds listed
in Table 1 is approximately 0.35 pKa units, with the
results for some compounds varying by greater than
0.9 pKa units (e.g., betahistine, chloroquine, levallorphan, and sulfasalazine).
The overall average difference between the
pKa values measured in this work to the average
literature pKa values was found to be 0.04 U
while the absolute average error was 0.16, indicating a very good level of agreement to previously
reported results when considering the variables
discussed above.
Figure 1 shows the correlation between the pKa
values measured in this work to the average of
available literature pKa values. Eight pKa values
were excluded from the correlation as we were
unable to ultimately confirm or identify a suitable
literature reference. For example, we found only
one pKa value reported for buspirone, cefuroxime
and histamine while we observed two pKa values
by our measurement. Kaufman et al.60 predicted
two pKa values for levallorphan, but they were
able to measure only one due to a decreasing
solubility of the compound. We were unable to
identify any reliable literature pKa values for
nafronyl and norephedrine.
The correlation of Figure 1 yielded a slope of
0.997 with an intercept of þ0.059 (R2 ¼ 0.993)
DOI 10.1002/jps
MULTIPLEXED CE pKa DETERMINATION
11
Figure 1. Correlation of aqueous pKa values measured by multiplexed 96-capillary
array CE with the average of pKa values reported in the literature.
providing further validation of the multiplexed
CE method for pKa measurement. It should be
noted that the correlation is somewhat affected by
the results obtained at the pH extremes, where
measurements are inherently more difficult by
any method due to the presence of significant
amounts of Hþ or OH species. For example, the
lower pKa values reported for sulfacetamide in the
literature were 1.76, 1.95, and <2.5 as compared
to our average result of 1.26. This data point,
being the lowest in the set, does have some effect
on the correlation. However, its removal yields
a slope of 1.000 with an intercept of þ0.037
(R2 ¼ 0.993) and thus the variation is of little
practical significance.
Interlaboratory Comparisons of pKa Results
To evaluate the interlaboratory reproducibility of
the multiplexed CE method for pKa measurement
we examined compounds within the test set which
were analyzed in both laboratories (Tab. 2, n ¼ 30
compounds, 53 pKa values). Good overall agreement was obtained between the two laboratories
considering the slight differences in instrument
design, capillary length, and experimental parameters. The average difference between measured pKa values was 0.02 U (0.12 average absolute
error) with the majority of results within 0.15 pKa
units of each other. The average repeatability
(SD) for the cePRO 9600TM instrument was 0.07 U
DOI 10.1002/jps
compared to 0.12 U for the MCE 2000 instrument.
We note that in both cases, measurements were
acquired over several months or longer with at
least two different lots of aqueous buffers, as
opposed to repetitive measurements on the same
day, for which the SD is most often 0.05 U or less.
Upon closer examination of Tables 1 and 2 it can
be observed that often the largest discrepancies
between results occur for multiprotic compounds
with closely spaced pKa values (less than 2 pH
units apart). In particular, several compounds
varied considerably between laboratories and/or
possessed relatively high standard deviations,
namely chloroquine, indomethacin, levallorphan,
methotrexate, and tetracycline. We sought to
explore these differences and further investigate
the multiplexed CE method in several areas—in
regard to the resolution of closely spaced pKa
values, the ability to identify potential compound
degradation or impurities, and the lower limit
of compound solubility for performing aqueous
measurements. We then describe a method for
the pKa measurement of aqueous insoluble compounds using mixed methanol/water cosolvent
buffers and extrapolation to 0% cosolvent.
Resolution of Closely Spaced pKa Values
The measurement of closely spaced pKa values
less than 2 pH units apart requires careful
attention to experimental parameters along with
JOURNAL OF PHARMACEUTICAL SCIENCES
12
SHALAEVA ET AL.
Table 2. Interlaboratory Comparison of pKa Values Measured by 96-Capillary Array CE Using Aqueous Buffers
(See Experimental Section for Differences Between Methods)
Compound
1
Acyclovir
2
Betahistine
3
Chloroquine
4
5
6
7
Cimetidine
Codeine
Flumequine
Furosemide
8
9
10
Ibuprofen
Indomethacin
Ketoconazole
11
Labetalol
12
Levallorphan
13
14
Lidocaine
Methotrexate (amethopterin)
15
Morphine
16
Naloxone
17
Norfloxacin
18
19
Papaverine
Piroxicam
20
21
Propranolol
Quinine
22
Sotalol
23
Sulpiride
24
Terbutaline
25
Tetracaine
26
Tetracycline
27
28
Trimipramine
Tripelennamine
39
Tyrosine
30
Warfarin
JOURNAL OF PHARMACEUTICAL SCIENCES
Average pKa
cePRO
SD
Average
pKa MCE 2000
SD
Difference
cePRO–MCE2000
2.20
9.20
3.88
9.98
7.27
10.64
6.93
8.14
6.29
3.60
10.39
4.34
4.01
3.13
6.35
7.35
9.17
9.22
10.57
7.97
3.27
3.53
5.15
8.13
9.31
8.01
9.13
6.18
8.37
6.38
1.87
5.34
9.59
4.33
8.49
8.31
9.59
9.01
10.09
8.79
9.60
2.25
8.51
3.36
7.09
9.30
9.51
4.07
9.09
2.23
8.85
10.06
4.94
0.03
0.01
0.02
0.06
0.09
0.07
0.06
0.06
0.05
0.06
0.05
0.06
0.09
0.07
0.05
0.03
0.04
0.02
0.15
0.06
0.07
0.11
0.04
0.05
0.06
0.04
0.03
0.04
0.05
0.05
0.06
0.06
0.05
0.07
0.07
0.03
0.01
0.03
0.05
0.02
0.02
0.05
0.04
0.09
0.88
0.23
0.02
0.05
0.00
0.02
0.06
0.06
0.03
2.20
9.17
3.93
10.07
7.77
10.75
6.85
8.18
6.34
3.59
10.31
4.37
4.36
3.25
6.25
7.36
9.14
9.29
10.86
7.95
3.36
4.30
5.51
7.99
9.07
8.15
8.96
6.12
8.26
6.27
1.74
5.21
9.58
4.24
8.57
8.45
9.35
9.08
10.11
8.79
9.46
2.34
8.49
3.36
7.10
9.00
9.61
4.10
9.10
2.11
8.69
10.01
5.02
0.07
0.01
0.11
0.14
0.10
0.03
0.01
0.15
0.06
0.05
0.04
0.07
0.31
0.19
0.12
0.05
0.19
0.05
0.29
0.03
0.08
0.38
0.15
0.13
0.04
0.09
0.21
0.06
0.01
0.06
0.16
0.04
0.16
0.07
0.05
0.05
0.08
0.07
0.05
0.10
0.28
0.10
0.08
0.05
0.69
0.48
0.16
0.07
0.04
0.04
0.09
0.14
0.14
0.00
0.03
0.05
0.09
0.50
0.11
0.08
0.04
0.05
0.01
0.08
0.03
0.35
0.12
0.10
0.01
0.03
0.06
0.29
0.02
0.09
0.77
0.36
0.14
0.24
0.14
0.17
0.06
0.11
0.11
0.13
0.13
0.01
0.09
0.08
0.14
0.24
0.07
0.02
0.00
0.14
0.09
0.02
0.00
0.01
0.30
0.10
0.03
0.01
0.13
0.16
0.05
0.08
DOI 10.1002/jps
MULTIPLEXED CE pKa DETERMINATION
data analysis and interpretation. For potentiometry, the sample should be fully solubilized and
of known concentration to accurately determine
the number of bound protons per molecule of
sample and to identify closely spaced pKa values
by means of a Bjerrum plot.5 In addition, the finite
electrode response time results in relatively long
analysis times when sampling at many different
pH points during the titration. When employing
UV spectrophotometric methods, difficulties arise
from overlapping spectra less than two pKa units
apart. In such cases, target factor analysis (TFA)
is required to deconvolute the results. It was
recommended to predict in advance how many
pKas are expected and their approximate values
since at times TFA can fit the same data for two,
three, or four pKa values.35
When measuring closely spaced pKa values by
CE it is important for the buffers used to be of
known pH value, evenly spaced, and of equal ionic
strength throughout the pH range. It has been
previously suggested that five pH points should be
used per one pKa value measured.61 The majority
of previously reported CE methods used 10–12
evenly spaced buffers to cover the pH range from 2
to 11, translating to a spacing of 0.8–0.9 pH units.
This spacing can impact the accurate measurement of pKa values closer than approximately
3 pH units apart. Increasing the number of buffers
is desirable when analyzing compounds with
multiple pKa values as shown by Ishihama
et al.,22 who measured up to six or seven ionizable
groups employing CE with 19 evenly spaced
buffers from pH 2 to 12.
The increase in throughput afforded by multiplexed CE in combination with the above considerations led us to develop a 24 point buffer
system covering the pH range from 1.7 to 11.2,
decreasing the spacing to 0.4 pH units. Even
spacing of the buffers was maintained to maximize the precision of measurements across the
entire pH range. In addition, we noticed that the
reproducibility suffered if the pH values between
batches were not exactly reproduced, especially
when measuring multiprotic compounds with
the pKa values less than 3 U apart.
Another factor influencing the measurement
of closely spaced pKa values is the separation
resolution between the compound and neutral
marker. To improve the separation resolution, the
vacuum level applied to the capillary array in this
work was minimized to yield a separation time of
10–13 min, as compared to previous CE studies
where separation times on the order of 1–5 min
DOI 10.1002/jps
13
were obtained by the use of high pressures.
Separation resolution is particularly important
when close pKa values are of different types as
in acid/base zwitterions, where the meff changes
from positive to negative causing inversion of
the migration order surrounding the compound
pI value. Examples of zwitterionic compounds
with closely spaced pKa values measured in this
study include labetalol, methotrexate, morphine,
naloxone, sotalol, and sulpiride. From Table 2, the
interlaboratory pKa differences for most multiprotic zwitterions were within 0.2 pKa units. The
largest discrepancy was for methotrexate, which
has three pKa values within two pH units of each
other. The differences in results were most likely
due to subtle variations in the buffer pH values
and/or slight differences in separation resolution
caused by differences in capillary length and
applied voltage between labs.
Importantly, the use of a previously developed
empirical relationship between compound MW,
meff and charge to predict the number of pKa
values present is vital when analyzing compounds
with closely spaced and/or unknown pKa values.28
In the case of methotrexate, a predicted charge of
þ0.95 and 1.76 indicated that the compound was
a monobase/diacid with three pKa values. Similarly, three pKa values were reported by Miller
et al.28 using the aforementioned empirical
relationship and by Bergström et al.62 using UV
spectrophotometry. However, only two pKa values
were reported by Wan et al.43 using CE-MS and
10 widely spaced pH buffers. This example
highlights the need to use meff and MW as an
indicator of compound charge when measuring pKa values by CE-based methods.
In some cases only partial resolution was
obtained between the compound and neutral
marker at pH points near the pI (isoelectric point)
value making the assignment of migration times
approximate at best. In a few other cases, an
additional third peak was observed near the pI
value of zwitterionic compounds, as shown in
Figure 2A for sulpiride at pH 9.66. Interestingly,
only two peaks were observed at pH values below
9.6 and above 10.0, with relative peak areas
within 1–2% of each other. Analysis by single
capillary CE with diode array UV detection at pH
9.6 found that the middle peak corresponded to
DMSO, while the leading and trailing peaks
possessed very similar UV spectra. When fitting
the meff versus pH data, the points at pH 9.6 and
10.0 noticeably diverged from the best-fit line
applied to the data (Fig. 2B). Removal of these
JOURNAL OF PHARMACEUTICAL SCIENCES
14
SHALAEVA ET AL.
Figure 2. (A) Overlay of electropherograms obtained at different pH values for 50 ppm
sulpride in 1 mM HCl, 0.1% DMSO. The peak assigned to DMSO is marked with . The
electropherograms are offset for clarity. (B) Plot of meff versus pH for sulpiride, assuming
the leading peak at pH 9.66 and the trailing peak at 10.02 correspond to sulpiride.
outlier points led to a better R2 value, increased
reproducibility and improved agreement to literature values. Although still under investigation, we speculate from the above findings that the
additional third peak observed at pH 9.6 may be
related to the presence of multiple stabilized
species with slightly different overall charge.
In summary, to maximize the measurement
precision of closely spaced pKa values by CE it
is important to utilize evenly spaced buffers
of carefully controlled pH and to ensure good
separation resolution between the compound and
neutral marker whenever possible, also by way of
adjusting the vacuum setting.
JOURNAL OF PHARMACEUTICAL SCIENCES
Identification of Compound Degradation/Impurities
and Solubility Limits for Aqueous pKa Measurements
A key advantage of CE when measuring
compound pKa values is the ability to separate
sample components possessing different mass-tocharge ratios. This ability is of substantial benefit
when working in a Pharmaceutical Discovery
setting, where the purity and stability of compounds has often not been fully assessed. A few
reports have described the application of CEbased pKa measurements to impure and/or labile
compounds. Örnskov et al.30 proposed a strategy
for measuring the pKa values for labile compounds
DOI 10.1002/jps
MULTIPLEXED CE pKa DETERMINATION
by CE including the use of a stabilizing sample
diluent, electrokinetic sample injection, rapid
analysis times, and characterization of peak
components by UV-Vis spectra. In addition to
measuring the pKa value, Simplı́cio et al.32 were
able to examine elimination rates of labile
compounds by CE. The integration of MS detection was also found to be useful when observing
compound impurities.43
When employing multiplexed CE for pKa
measurement, a single fixed UV wavelength is
employed for detection and therefore identification of sample components by UV-Vis or mass
spectra is not possible. However, degradation of
labile compounds can often be assessed by differences in measured meff and migration behavior
as shown in Figure 3 for indomethacin, a wellknown case used as an example here. Three
distinct analyte peaks were observed over the
majority of pH values above pH 3.9 for indomethacin samples prepared in 1 mM NaOH
(Fig. 3). Plots of meff versus pH could be derived
for the three distinct species and the measured
apparent pKa values (I ¼ 50 mM) and predicted
charge valency for the three species (assuming a
MW of 358 for intact indomethacin) were 4.26
(1.04), 4.35 (1.30), and 3.89 (1.64).
The intact compound should have the highest
MW and therefore the lowest meff indicating that
the leading analyte peak in Figure 3 is indomethacin and trailing two analyte peaks are
degradation products. This is indeed the case, as
analysis of indomethacin prepared in water yields
a single analyte peak corresponding to the leading
15
peak with an apparent pKa value and predicted
valency of 4.04 (1.06). The differences in
measured pKa values between indomethacin
prepared in water and 1 mM NaOH are due to
partial peak overlap with the sample degradants
at pH values near the pKa value. Importantly,
the ability to predict compound valency permitted
the identification of the intact compound in the
presence of multiple degradation impurities, and
all species in this case could be separated and
their pKa values measured by CE. Such an
analysis is not possible by traditional batch
methods employing potentiometry or spectrophotometry.
The solubility limitations of many drug-like
compounds also deserve some comments as we
tested a number of compounds previously identified as low or sparingly soluble (e.g., amiodarone,
tamoxifen, indomethacin, flumequine, furosemide, ketoconazole) 35,43,62 and found that a large
number were easily measurable by our method
using aqueous buffers. However, some compounds
(identified by superscript a in Tab. 1) presented a
challenge when analyzed at typical working
concentrations of 50–100 mg/mL. Precipitation
of low solubility compounds when employing CE
for pKa measurement is identifiable by significant
broadening of analyte peaks or the appearance
of a plateau in place of the typical Gaussian
peak profile obtained for compounds in solution.
Another indication of precipitation is an abrupt
disappearance of the analyte peak at a particular
pH value. The precipitation of a low solubility
compound is shown in Figure 4A for the case of
Figure 3. Overlay of electropherograms obtained for 50 ppm indomethacin at pH
10.40, dissolved in different sample matrices containing 0.1% DMSO neutral marker
(marked by ).
DOI 10.1002/jps
JOURNAL OF PHARMACEUTICAL SCIENCES
16
SHALAEVA ET AL.
Figure 4. (A) Overlay of electropherograms obtained at pH 9.20 and pH 9.60 for
amitriptyline prepared at 50 ppm concentration (top two traces) and diluted 10-fold to
5 ppm concentration (bottom two traces), containing 0.1% DMSO neutral marker (). The
electropherograms are offset for clarity. (B) Plots of meff versus pH and best-fit lines for
amitriptyline analyzed at 50 ppm concentration (&) and diluted 10-fold to 5 ppm
concentration (~). Precipitation occurring at pH 9.20 and above in the more concentrated sample resulted in an underestimation of the pKa value.
amitriptyline, which possesses a reported aqueous
intrinsic solubility of 1.8 mg/mL (6.5 mM).5 The
result is a general underestimation of pKa and
dilution or use of cosolvent are required to solve
the problem, as shown still for amitriptyline in
Figure 4B, but its assessment a priori is difficult
even with log D data.
Potential Challenges with Aqueous
pKa Measurements
In addition to solubility issues encountered during
aqueous pKa analysis, a few compounds exhibited
JOURNAL OF PHARMACEUTICAL SCIENCES
a particular behavior during measurement, for
example chloroquine and tetracycline in certain
pH ranges, and yielding broad peaks.
The anomalous results for chloroquine and
tetracycline may be due either to an interaction
with silanol groups on the capillary wall or with
the phosphate buffer ions. It has been reported
that tetracycline has a strong affinity for silanol
groups present in HPLC stationary phases.63
Additionally, interactions between certain multiply charged and/or hydrophobic basic compounds
with the negatively charged capillary wall has
been previously discussed.64 In these cases alterDOI 10.1002/jps
MULTIPLEXED CE pKa DETERMINATION
native buffers have been previously utilized for
CE-based pKa measurements, including MES,
HEPES, MOPS, TAPS, AMPSO, CAPS, etc.21,22 In
addition, certain amine containing buffers such as
MES and HEPES are known to interact with the
capillary walls, effectively competing with and
diminishing analyte adsorption.64
Methanol/Water Cosolvent pKa Measurements
Comparison of Cosolvent pKa Measurements
to Literature Values
Several compounds in the test set (e.g., amiodarone and chlorpromazine) were found to precipitate
during aqueous measurements, even when diluted to the detection limits of the more sensitive
cePRO instrument (5–10 mg/mL, corresponding
to 17–34 mM for MW ¼ 300). To measure the
pKa values for compounds with low aqueous
17
solubility a method employing methanol/water
cosolvent buffers was developed. The apparent
s
w pKa values (I ¼ 50 mM) were measured over four
decreasing percentages of methanol cosolvent
(60%, 50%, 40%, 30%, v/v), converted to apparent
s
s pKa values using the appropriate d term via
Eq. (3), and extrapolated to 0% cosolvent to yield
the apparent w
w pKa value using the widely
accepted Y–S method.9,56,57,59 In addition to the
aforementioned low solubility compounds, the
data set used to evaluate the cosolvent method
included 16 additional compounds previously
measured with aqueous buffers to further assess
the approach. The data set consisted primarily
of monobasic compounds with two dibasic compounds (quinacrine and quinine), one acidic
compound (flufenamic acid) and one monoacidic/
monobasic zwitterion (mebendazole).
Table 3 lists the average extrapolated apparent w
w pKa values at I ¼ 50 mM measured by
Table 3. Summary of pKa Values Measured by 96-Capillary Array CE Using Mixed Methanol/Water Buffers with
Yasuda–Shedlovsky Extrapolation to 0% Cosolvent and a Comparison to Literature Values
Compound
a
1
2
3
4
5
6
7
8
9
10
Amiodarone
Amitriptyline
Bifonazole
Chlorpromazine
Clomipramine
Clotrimazole
Desipramine
Flufenamic Acid
Imipramine
Mebendazole
11
12
13
14
15
16
17
Miconazole
Nefazodone
Nicardipine
Nortriptyline
Promazine
Promethazine
Quinacrine
18 Quinine
19
20
21
22
23
Tamoxifenb
Terfenadineb
Trazodone
Trimipramine
Verapamil
pKa Average, pKa Average,
pKa Range
This Work
Literature Difference Literature
8.71
9.49
6.11
9.16
9.36
5.83
10.30
4.01
9.50
3.20
9.64
6.38
6.65
7.12
10.02
9.38
8.71
7.29
9.87
4.39
8.53
8.60
9.40
6.60
9.25
8.50
8.88
9.41
5.80
9.25
9.28
5.89
10.27
4.02
9.50
3.43
9.93
6.35
n/a
7.34
10.11
9.11
8.93
7.74
10.08
4.20
8.51
8.60
9.54
6.75
9.25
8.81
0.17
0.08
0.31
0.09
0.08
0.06
0.03
0.01
0.00
0.23
0.29
0.03
0.22
0.09
0.27
0.22
0.45
0.21
0.19
0.02
0.00
0.14
0.15
0.00
0.31
References
8.7–9.06
5,73
9.32–9.49
5,45,51,71,72
5.72–5.88
77 c
9.15–9.38
43,67,68,69,109,121
9.17–9.38
71 c
5.48–6.3
85 c
10.16–10.65
5,35,51,72,74,86
3.63–4.27
5,27,33,67
9.34–9.66
5,34,35,43,49,51,72 c
3.43
43
9.93
6.07–6.63
5,28
n/a
7.17–7.41
28,109,122
10.02–10.19
5,45,49,51
8.92–9.40
72,109,121
8.62–9.10
5,68,72,109
7.73–7.74
68,123
9.97–10.18
3.95–4.46 13,14,21,26,28,29,33,35,43,45,59,68,82,91
8.35–8.60
8.48–8.71
5,43
9.21–9.86
5,43
6.69–6.79
33,49 c
9.15–9.37
49 cd
8.66–9.07
5,22,27,28,35,58,74,109
a
Due to precipitation, only values from 50% to 60% (v/v) methanol were used.
Due to precipitation, only values from 40% to 60% (v/v) methanol were used.
c
Potentiometric measurement performed by pION, Inc.
d
Potentiometric measurement Pfizer, Sandwich Laboratories.
b
DOI 10.1002/jps
JOURNAL OF PHARMACEUTICAL SCIENCES
18
SHALAEVA ET AL.
multiplexed CE using the cosolvent method with
the Y–S extrapolation along with a comparison
to published literature pKa data. Literature data
were treated in a similar manner as the
aqueous pKa measurement comparisons. The
majority of literature data for the relatively low
solubility compounds in the data set were
obtained using Y–S cosolvent extrapolation methods. The average difference between the cosolvent
extrapolated pKa values in this work and the
average literature pKa values was found to be
0.07 U (average absolute error of 0.15 U). A
correlation plot of the data from Table 3 yielded
a slope of 1.005 with an intercept of þ0.027
(R2 ¼ 0.992) (data not shown). Taken together,
these results indicate a good level of agreement
between the cosolvent method of this work
compared to previous studies.
We note that it was not possible to analyze a few
compounds over the entire cosolvent range from
30% (v/v) to 60% (v/v) methanol due to precipitation during the analysis. Tamoxifen and terfenadine were found to precipitate at 30% (v/v)
methanol, while amiodarone precipitated up to
40% (v/v) methanol. With a reported aqueous
intrinsic solubility of only 0.005 mg/mL (8 nM),5
amiodarone represents an extreme lower limit
of aqueous solubility and yet its pKa could be
determined over at least two percentages of
methanol cosolvent.
Figure 5 shows some representative Y–S extrapolation plots of 1/e (1000) versus (apparent
s
s pKa þ log[H2O]) for several different compounds.
The plots display good linearity (R2 0.97, except
for the upper pKa of mebendazole where R2 ¼ 0.75)
and varying slopes depending upon the compound
structure and ionization type, with an observed
trend of acidic compounds showing a positive slope
and basic compounds showing a negative slope,
consistent as well with earlier reports which
found that basic ionizable groups produce negative slopes while acidic groups possess positive
slopes when performing Y–S extrapolations from
methanol cosolvent mixtures.9,59 The decrease
in pKa value for bases and increase in pKa value
for acids with increasing methanol content results
from suppression of ionization upon the corresponding decrease in the dielectric constant of the
medium. The slopes for the Y–S plots in this work
varied somewhat between compounds depending
upon the type of ionizable group. A detailed
analysis of Y–S plot slope variations is beyond the
scope of this study, but has been previously
attributed to differences in both the chemical
and solvation structure of compounds.59
Interlaboratory Comparisons of
Cosolvent pKa Measurements
Table 4 shows a comparison between laboratories
of the extrapolated apparent w
w pKa values (I ¼
50 mM) obtained using the multiplexed CE
cosolvent method. The absolute average difference between measured pKa values was 0.12 U.
The average repeatability (SD) was comparable
between instruments (cePRO 0.06, MCE 2000 0.09). Again, in both cases measurements were
acquired over several months with at least two
differently prepared lots of cosolvent buffers to
better simulate long-term repeatability.
Figure 5. Representative Yasuda–Shedlovsky pKa extrapolation plots obtained for
various compounds.
JOURNAL OF PHARMACEUTICAL SCIENCES
DOI 10.1002/jps
MULTIPLEXED CE pKa DETERMINATION
19
Table 4. Comparison of pKa Values Measured by Two Laboratories/Instruments
Using Mixed Cosolvent Buffers
Compound
a
Amiodarone
Amitriptyline
Bifonazole
Chlorpromazine
Clomipramine
Clotrimazole
Desipramine
Flufenamic acid
Imipramine
Mebendazole
Miconazole
Nefazodone
Nicardipine
Nortriptyline
Promazine
Promethazine
Quinacrine
Quinine
Tamoxifenb
Terfenadineb
Trazodone
Trimipramine
Verapamil
pKa by cePRO
SD
8.78
0.08
6.18
9.21
9.35
5.87
0.02
0.04
0.05
0.03
4.01
9.50
3.20
9.64
6.40
6.67
7.26
10.03
9.47
8.71
7.29
9.98
4.41
8.64
8.62
9.52
6.67
9.29
8.68
0.04
0.09
0.06
0.14
0.06
0.05
0.02
0.06
0.03
0.13
0.06
0.12
0.12
0.05
0.10
0.05
0.03
0.08
0.04
pKa by
MCE2000
SD
8.64
9.49
6.01
9.10
9.37
5.78
10.30
0.15
0.16
0.03
0.07
0.12
0.09
0.20
9.51
0.03
0.01
6.30
6.63
6.98
10.00
9.32
8.71
0.11
0.01
0.07
0.04
0.08
0.03
0.10
0.04
0.28
0.03
0.15
0.00
9.75
4.36
8.42
8.57
9.27
6.52
9.16
8.35
0.16
0.13
0.05
0.19
0.08
0.08
0.05
0.09
0.23
0.05
0.22
0.05
0.25
0.15
0.13
0.33
Difference
0.14
0.17
0.11
0.02
0.09
a
Due to precipitation, only values from 50% to 60% (v/v) methanol were used.
Due to precipitation, only values from 40% to 60% (v/v) methanol were used.
b
From Table 4 it can be observed that the Y–S
extrapolated results from the MCE 2000 are in
general slightly lower than the results from the
more sensitive cePRO instrument. One possible
explanation for this result is the longer time to fill
the inlet pH buffers into the capillary array and
perform the analysis on the MCE 2000 instrument. The buffer plates used in both instruments
are not sealed to avoid damage to the capillary tips
which could result from the repetitive piercing of
well caps. In addition, in the case of the MCE 2000
instrument a few results were obtained from
the repeated use of the same prepared cosolvent
buffer plate. As a result, some evaporation likely
occurred from the buffer plate between analyses,
particularly at higher % methanol contents. Any
decrease in methanol concentration would lead
to an increase in measured apparent sw pKa values
for basic compounds, with greater evaporation
occurring at higher methanol content. Indeed,
a comparison of the results for nicardipine
DOI 10.1002/jps
and verapamil between labs revealed that the
average apparent sw pKa values at 60% (v/v)
methanol were higher by 0.11 and 0.07 U,
respectively, on the MCE 2000 instrument compared to the cePRO instrument. For this reason
the repetitive use of cosolvent buffer plates was
discontinued.
It was also observed in the course of this work
that a measurable increase in temperature occurs
during the titration to the target sw pH value when
preparing the cosolvent buffers. Upon cooling to
room temperature, pH shifts of up to 0.1 U were
observed for a few of the cosolvent buffers. The
preparation procedure has since been modified to
allow cooling of the solution to room temperature
prior to final buffer pH adjustment. This also may
account for some of the observed differences in
results.
The more automated cePRO instrument should
at any rate eliminate most of the instrumentrelated experimental variations due to manual
JOURNAL OF PHARMACEUTICAL SCIENCES
20
SHALAEVA ET AL.
sample/buffer plate exchange or different capillary filling and analysis times.
Comparisons of Aqueous and Cosolvent pKa Results
Table 5 shows a comparison between the average
extrapolated apparent w
w pKa values measured
with the multiplexed CE cosolvent method and
the average apparent pKa values obtained with
aqueous buffers. The average deviation of the Y–S
extrapolations from aqueous measurements for
basic pKa values was 0.17 U (n ¼ 14) and for
acidic pKa values was þ0.06 (n ¼ 2) with an overall absolute average error of 0.17. Although a
relatively small number of compounds were
compared, this result is again in agreement with
earlier studies which found that the Y–S extrapolation method in general yielded linear plots
with a slight underestimation of pKa values for
basic compounds and a slight overestimation
of pKa values for acidic compounds.59
Further Observations on Cosolvent
pKa Measurements
Overall, the multiplexed CE cosolvent method
described above was able to successfully measure
the pKa values for low solubility compounds with
good agreement to previous literature data. The
total time to measure the extrapolated w
w pKa
values for four compounds over 24 pH points
and four % cosolvent compositions was approximately 2 h. This is a significant improvement in
sample throughput over the traditional cosolvent
potentiometric method, which requires 2–3 h for
measurement of a single compound over 3–4%
cosolvent compositions, and single capillary CE
methods which would require many hours to
measure a single pKa value over several different
cosolvent compositions.
We found that it was important to prepare
the cosolvent buffer plates immediately prior to
performing the cosolvent experiments, in order to
minimize any effects of buffer evaporation on the
experiment. Using a repeater pipette, it is possible
to prepare the 24 point buffer plates in approximately 10 min. This preparation time can be
eliminated through the use of premade, sealed
buffer plates which are currently under development. For the same reason the cosolvent buffer
plates should not be reused for multiple experiments.
We also observed that it is beneficial to prepare
samples for the cosolvent measurements in 60%
(v/v) methanol to better match the composition of
the buffer solutions. This is due to an improvement of the DMSO (the neutral marker employed
in this work) peak shape, non-Gaussian particularly at higher pH values and fully aqueous
diluents, which could affect the proper assignment of the migration time. The use of a sample
diluent which contains a similar or higher
Table 5. A Comparison Between the Average Extrapolated Apparent w
w pKa Values
Obtained with the Cosolvent Method via Y–S Extrapolation and the Average
Apparent pKa Values Obtained with Aqueous Buffers
Compound
Amitriptyline
Bifonazole
Clomipramine
Clotrimazole
Desipramine
Flufenamic acid
Imipramine
Mebendazole
Nortriptyline
Promethazine
Quinine
Trazodone
Trimipramine
Verapamil
JOURNAL OF PHARMACEUTICAL SCIENCES
pKa, Average,
Y–S Extrapolation
pKa, Average,
Aqueous
Difference
9.49
6.10
9.36
5.83
10.30
4.02
9.51
3.20
9.64
10.02
8.71
4.39
8.53
6.52
9.23
8.52
9.51
6.29
9.57
5.99
10.42
3.97
9.58
3.27
9.56
10.29
9.05
4.29
8.53
6.80
9.56
8.93
0.02
0.19
0.21
0.16
0.12
0.05
0.07
0.07
0.08
0.27
0.34
0.10
0.00
0.28
0.33
0.41
DOI 10.1002/jps
MULTIPLEXED CE pKa DETERMINATION
proportion of methanol compared to the run buffer
greatly improved the DMSO peak shape.
When analyzing the cosolvent titration curve
data for several basic compounds with relatively
high pKa values (e.g., clomipramine, nortriptyline, imipramine) there was a noticeable ‘‘jump’’
in the effective mobility upon increasing the
s
w pH value and switching between phosphate
(sw pH ¼ 8.40) and borate (sw pH ¼ 8.80) cosolvent
buffers. This result is consistent with a recent
report by de Nogales and coworkers who observed
that the measured effective mobility was lower for
several hydrophobic basic drugs when 50% (v/v)
methanol/water cosolvent buffers were prepared
from phosphate and/or borate as compared to
other common buffers such as tris, ammonia,
butylamine, or ethanolamine.65 In the case of
phosphate, this phenomenon was ascribed to
specific interactions taking place between the
negatively charged phosphate buffer ions and
the positively charged analyte. Comparison of the
measured apparent ss pKa values (I ¼ 50 mM) for
amiodarone, trimipramine, imipramine, and nortriptyline at 50% (v/v) methanol–water to values
measured by de Nogales and coworkers found that
on average the values measured in this work are
approximately 0.1 pH units lower, indicating that
the use of phosphate and/or borate buffers may
have resulted in a slight underestimation of the
measured pKa values. Further work is needed to
more fully elucidate the effects of buffer type on
the measured mobility of different compounds in
cosolvent mixtures. A potentially useful application of this observation is that it may provide a
tool to directly identify potential buffer-analyte
interactions and their effect on the measured
pKa value.
CONCLUSION
We have reported a good and fairly rapid method
for the measurement of pKa values by multiplexed
CE which has been shown to yield accurate results
with low inter- and intra-laboratory variability.
The method is capable of handling measurements
made using water soluble compounds and low
solubility compounds alike, via the use of a cosolvent approach we have optimized and adopted
after extensive testing.
Our stated goals included the study of the
aspects mentioned above and, among others, the
use of an enhanced set of buffers to be able to
discern closely spaced pKa values, often missed by
DOI 10.1002/jps
21
other types of measurements and, in some cases,
reported as such in the literature. We have
discussed prior efforts and used quality literature
data, from thorough searches and data analysis,
to benchmark our results. We have kept in mind
all along the application of the method in an
industrial discovery setting and offered practical
considerations and clues, based on extensive
experience with the method by two laboratories,
on issues one may encounter with stability and
low solubility of compounds and how to recognize
and overcome some of them. We have also shown
that the use of mass spectrometric detection is not
warranted in most cases as we were able to
successfully measure extremely low solubility
compounds.
We believe this method is readily applicable and
capable of yielding accurate results and also offers
added advantages in terms of the low amount of
compound needed. Importantly, the multiplexed
CE method is capable of recognizing the number
of pKa values present in a compound by relating
compound MW, effective mobility, and charge.
Finally we also note, among its potential advantages over other methods, the possibility to
identify potential solubility and/or stability and/
or buffer-solute interaction issues in addition to
measuring the pKa value.
REFERENCES
1. Lin J, Sahakian DC, Morais SMd, Xu JJ, Polzer RJ,
Winter SM. 2003. The role of absorption, distibution, metabolism, excretion and toxicity in drug
discovery. Curr Top Med Chem 3:1125–1154.
2. Lombardo F, Obach RS, Shalaeva MY, Gao F. 2004.
Prediction of human volume of distribution values
for neutral and basic drugs. 2. Extended data set
and leave-class-out statistics. J Med Chem 47:
1242–1250.
3. Kerns EH. 2001. High throughput physicochemical
profiling for drug discovery. J Pharm Sci 90:1838–
1858.
4. Avdeef A, Testa B. 2002. Physicochemical profiling
in drug research: A brief survey of the state-of-theart experimental techniques. Cell Mol Life Sci 59:
1681–1689.
5. Avdeef A. 2003. Absorption and drug development:
Solubility, permeability, and charge state. Hoboken, NJ: John Wiley & Sons.
6. Comer JEA. 2003 High-throughput measurements
of log D and pKa. In: Waterbeemd Hvd, Lennernäs
H, Artursson PV, editors. Drug bioavailability, Vol.
18. Weinheim: Wiley-VCH. pp 21–45. [Mannhold R,
JOURNAL OF PHARMACEUTICAL SCIENCES
22
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
SHALAEVA ET AL.
Kubinyi H, Folkers G (Series Editor): Methods and
principles in medicinal chemistry, vol 18].
Avdeef A, Bucher JJ. 1978. Accurate measurements
of the concentration of hydrogen ions with a glass
electrode: Calibrations using the Prideaux and
other universal buffer solutions and a computercontrolled automatic titrator. Anal Chem 50: 2137–
2142.
Albert A, Serjeant EP. 1984. The determination of
ionization constants, 3rd edition. New York: Chapman and Hall.
Avdeef A, Comer JEA, Thomson SJ. 1993. pHmetric log P. 3. Glass electrode calibration in methanol-water, applied to pKa determination of waterinsoluble substances. Anal Chem 65:42–49.
Comer JEA, Avdeef A, Box KJ. 1995. Limits for
successful measurement of pKa and log P by pHmetric titration. Am Lab 4:36C–36I.
Gampp H, Maeder M, Meyer CJ, Zuberbuhler AD.
1985. Calculation of equilibrium constants from
multiwavelength spectroscopic data. I. Mathematical considerations. Talanta 32:95–101.
Seok Y-J, Yang K-S, Kang S-O. 1995. A simple
spectrophotometric determination of dissociation
constants of organic compounds. Anal Chim Acta
306:351–356.
Allen RI, Box KJ, Comer JEA, Peake C, Tam KY.
1998. Multiwavelength spectrophotometric determination of acid dissociation constants of ionizable
drugs. J Pharm Biomed Anal 17:699–712.
Tam KY, Takacs-Novak K. 2001. Multi-wavelength
spectrophotometric determination of acid dissociation constants: A validation study. Anal Chim Acta
434:157–167.
Ando HY, Heimbach T. 1997. pKa determinations
by using a HPLC equipped with DAD as a flow
injection apparatus. J Pharm Biomed Anal 16:
31–37.
Manderscheid M, Eichinger T. 2003. Determination of pKa values by liquid chromatography.
J Chromatogr Sci 41:323–326.
Roses M. 2004. Determination of the pH of binary
mobile phases for reversed-phase liquid chromatography. J Chromatogr A 1037:283–298.
Beckers JL, Everaerts FM, Ackermans MT. 1991.
Determination of absolute mobilities, pK values
and separation numbers by capillary zone electrophoresis. Effective mobility as a parameter for
screening. J Chromatogr A 537:407–428.
Cai J, Smith JT, Rassi ZE. 1992. Determination of
the ionization constants of weak electrolytes by
capillary zone electrophoresis. J High Resolut Chromatogr 15:30–32.
Cleveland JA, Benko MH, Gluck SJ, Walbroehl YM.
1993. Automated pKa determination at low
solute concentrations by capillary electrophoresis.
J Chromatogr A 652:301–308.
JOURNAL OF PHARMACEUTICAL SCIENCES
21. Gluck SJ, Cleveland JA. 1994. Capillary zone electrophoresis for the determination of dissociation
constants. J Chromatogr A 680:43–48.
22. Ishihama Y, Oda Y, Asakawa N. 1994. Microscale
determination of dissociation constants of multivalent pharmaceuticals by capillary electrophoresis. J Pharm Sci 83:1500–1507.
23. Mrestani Y, Neubert R, Munk A, Wiese M. 1998.
Determination of dissociation constants of cephalosporins by capillary zone electrophoresis. J Chromatogr A 803:273–278.
24. Barbosa J, Barron D, Jimenez-Lazano E. 1999.
Electrophoretic behaviour of quinolines in capillary
electrophoresis. Effect of pH and evaluation
of ionization constants. J Chromatogr A 839:183–
192.
25. Caliaro GA, Herbots CA. 2001. Determination
of pKa values of basic new drug substances
by CE. J Pharm Biomed Anal 26:427–434.
26. Jia Z, Ramstad T, Zhong M. 2001. Mediumthroughput pKa screening of pharmaceuticals by
pressure-assisted capillary electrophoresis. Electrophoresis 22:1112–1118.
27. Ishihama Y, Nakamura M, Miwa T, Kajima
T, Asakawa N. 2002. A rapid method for pKa determination of drugs using pressure-assisted capillary
electrophoresis with photodiode array detection in
drug discovery. J Pharm Sci 91: 933–942.
28. Miller JM, Blackburn AC, Shi Y, Melzak AJ, Ando
HY. 2002. Semi-empirical relationships between
effective mobility, charge, and molecular weight
of pharmaceuticals by pressure-assisted capillary
electrophoresis: Applications in drug discovery.
Electrophoresis 23:2833–2841.
29. Wan H, Holmen A, Nagard M, Lindberg W. 2002.
Rapid screening of pKa values of pharmaceuticals
by pressure-assisted capillary electrophoresis combined with short-end injection. J Chromatogr A
979:369–377.
30. Ornskov E, Linusson A, Folestad S. 2003. Determination of dissociation constants of labile drug compounds by capillary electrophoresis. J Pharm
Biomed Anal 33:379–391.
31. Foulon C, Danel C, Vaccher C, Yous S, Bonte J-P,
Goossens J-F. 2004. Determination of ionization
constants of N-imidazole derivatives, aromatase
inhibitors, using capillary electrophoresis and
influence of substituents on pKa shifts. J Chromatogr A 1035:131–136.
32. Simplicio AL, Gilmer JR, Frankish N, Sheridan H,
Walsh JJ, Clancy JM. 2004. Ionisation characteristics and elimination rates of some aminoindanones determined by capillary electrophoresis.
J Chromatogr A 1045:233–238.
33. Zhou C, Jin Y, Kenseth JR, Stella M, Wehmeyer
KR, Heineman WR. 2005. Rapid pKa estimation
using vacuum-assisted multiplexed capillary elec-
DOI 10.1002/jps
MULTIPLEXED CE pKa DETERMINATION
34.
35.
36.
37.
38.
39.
40.
41.
42.
43.
44.
45.
46.
47.
trophoresis (VAMCE) with ultraviolet detection.
J Pharm Sci 94:576–589.
Poole SK, Patel S, Dehring K, Workman H, Poole
CF. 2004. Determination of acid dissociation constants by capillary electrophoresis. J Chromatogr A
1037:445–454.
Box K, Bevan C, Comer J, Hill A, Allen R, Reynolds
D. 2003. High throughput measurement of pKa
values in a mixed-buffer linear pH gradient system.
Anal Chem 75:883–892.
Weinberger R. 2005. Determination of the pKa of
small molecules by capillary electrophoresis.
Am Lab 37:36–38.
Jia Z. 2005. Physicochemical profiling by capillary
electrophoresis. Curr Pharm Anal 1:41–56.
Wan H, Ulander J. 2006. High-throughput pKa
screening and prediction amenable for ADME profiling. Expert Opin Drug Metab Toxicol 2: 139–155.
Gong X, Yeung ES. 1999. An absorption detection
approach for multiplexed capillary electrophoresis
using a linear photodiode array. Anal Chem 71:
4989–4996.
Pang H-m, Kenseth J, Coldiron S. 2004. Highthroughput multiplexed capillary electrophoresis
in drug discovery. Drug Discov Today 9:1072–1080.
Lipinski CA, Lombardo F, Dominy BW, Feeney PJ.
1997. Experimental and computational approaches
to estimate solubility and permeability in drug
discovery and development settings. Adv Drug
Deliv Rev 23:3–25.
Kane RS, Glink PT, Chapman RG, MacDonald JC,
Jensen PK, Gao H, Pasa-Tolic L, Smith RD, Whitesides GM. 2001. Basicity of the amino groups of the
aminoglycoside amikacin using capillary electrophoresis and coupled CE-MS-MS techniques. Anal
Chem 73:4028–4036.
Wan H, Holmen AG, Wang Y, Lindberg W, Englund
M, Nagard MB, Thompson RA. 2003. Highthroughput screening of pKa values of pharmaceuticals by pressure-assisted capillary electrophoresis
and mass spectrometry. Rapid Commun Mass Spectrom 17:2639–2648.
Avdeef A, Box KJ, Comer JEA, Gilges M, Hadley M,
Hibbert C, Patterson W, Tam KY. 1999. pH-metric
log P 11. pKa determination of water-insoluble
drugs in organic solvent-water mixtures. J Pharm
Biomed Anal 20:631–641.
Buckenmaier SMC, McCalley DV, Euerby MR.
2004. Determination of ionisation constants of
organic bases in aqueous methanol solutions using
capillary electrophoresis. J Chromatogr A 1026:
251–259.
Compendium of analytical nomeclature, Definitive
rules 1997. on World Wide Web URL http://www.
iupac.org/publications/analytical_compendium/
Canals I, Oumada FZ, Roses M, Bosch E. 2001.
Retention of ionizable compounds on HPLC. 6.
pH measurements with the glass electrode in
DOI 10.1002/jps
48.
49.
50.
51.
52.
53.
54.
55.
56.
57.
58.
59.
60.
61.
23
methanol-water mixtures. J Chromatogr A 911:
191–202.
Roses M, Bosch E. 2002. Influence of mobile phase
acid-base equilibria on the chromatographic behaviour of protolytic compounds. J Chromatogr A
982:1–30.
Ruiz R, Rafols C, Roses M, Bosch E. 2003.
A potentially simpler approach to measure
aqueous pKa of insoluble basic drugs containing
amino groups. J Pharm Sci 92:14731481.
Porras SP, Riekkola M-L, Kenndler E. 2001. Capillary zone electrophoresis of basic analytes in
methanol as non-aqueous solvent. Mobility and
ionisation constant. J Chromatogr A 905:259–268.
Cantu MD, Hillebrand S, Carrilho E. 2003. Determination of the dissociation constants (pKa) of secondary and tertiary amines in organic media by
capillary electrophoresis and their role in the electrophoretic mobility order inversion. J Chromatogr
A 1068:99–105.
Psurek A, Scriba GKE. 2003. Peptide separations
and dissociation constants in nonaqueous capillary
electrophoresis: Comparison of methanol and aqueous buffers. Electrophoresis 24:765–773.
Barron D, Jimenez-Lozano E, Barbosa J. 2001.
Prediction of electrophoretic behaviour of a series
of quinolines in aqueous methanol. J Chromatogr A
919:395–406.
Jimenez-Lozano E, Marques I, Barron D, Beltran
JL, Barbosa J. 2002. Determination of pKa values of
quinolines from mobility and spectroscopic data
obtained by capillary electrophoresis and a diode
array detector. Anal Chim Acta 464:37–45.
Bellini S, Uhrova M, Deyl Z. 1997. Determination of
the thermodynamic equilibrium constants of waterinsoluble (sparingly soluble) compounds by capillary electrophoresis. J Chromatogr A 772:91–101.
Yasuda M. 1959. Dissociation constants of some
carboxylic acids in mixed aqueous solvents. Bull
Chem Soc Jpn 32:429–432.
Shedlovsky T. 1962. The behaviour of carboxylic
acids in mixed solvents. In: Pesce B, editor. Electrolytes. New York: Pergamon Press. pp 146–151.
Hasegawa J, Fujita T, Hayashi Y, Iwamoto K,
Watanabe J. 1984. pKa determination of verapamil
by liquid-liquid partition. J Pharm Sci 73:442–445.
Takács-Novák K, Box KJ, Avdeef A. 1997.
Potentiometric pKa determination of water-insoluble compounds: Validation study in methanol/water
mixtures. Int J Pharm 151:235–248.
Kaufman JJ, Semo NM, Koski WS. 1975 Microelectrometric titration measurement of the pKa’s and
partition and drug distribution coefficients of narcotics and narcotic antagonists and their pH and
temperature dependence. J Med Chem 18: 647–655.
Avdeef A. 1993. Application and theory guide to
pH-metric pKa and log P determination. Forest
Row, UK: Sirius Analytical Instruments Ltd.
JOURNAL OF PHARMACEUTICAL SCIENCES
24
SHALAEVA ET AL.
62. Bergström CAS, Strafford M, Lazorova L, Avdeef A,
Luthman K, Artursson P. 2003. Absorption classification of oral drugs based on molecular surface
properties. J Med Chem 46:558–570.
63. Oka H, Ito Y, Matsumoto H. 2000. Chromatographic analysis of tetracycline antibiotics in foods.
J Chromatogr A 882:109–133.
JOURNAL OF PHARMACEUTICAL SCIENCES
64. Righetti PG, editor. 1996. Capillary electrophoresis
in analytical biotechnology. New York: CRC Press.
65. de Nogales V, Ruiz R, Roses M, Rafols C, Bosch E.
2006. Background electrolytes in 50% methanol/
water for the determination of acidity constants
of basic drugs by capillary zone electrophoresis.
J Chromatogr A 1123:113–120.
DOI 10.1002/jps