Measurement of Dissociation Constants (pKa Values) of ... · Measurement of Dissociation Constants (pK a Values) of Organic Compounds by Multiplexed Capillary ... The acid–base

Measurement of Dissociation Constants (pKa Values) ofOrganic Compounds by Multiplexed CapillaryElectrophoresis Using Aqueous and Cosolvent Buffers

MARINA SHALAEVA,1 JEREMY KENSETH,2 FRANCO LOMBARDO,1 ANDREA BASTIN2

1Pfizer Global Research and Development, Groton Laboratories, Groton, Connecticut 06340

2CombiSep, 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

This article cwww.interscien

Advanced AnSep), 2711 Sout

Franco LombaBiomedical ReseMA 02139.

Corresponden4003; Fax: 617-com)

Journal of Pharm

� 2008 Wiley-Liss

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 arecompared to literature values obtained from multiple references. Two versions of theinstrument in two different labs were used to collect data over a period of 3 yearsand inter-laboratory and inter-instrument variations are discussed. Twenty-fourpoint aqueous and mixed cosolvent buffer systems were employed to improve theaccuracy of pKa measurements. It has been demonstrated that the method allowsdirect pKa measurements in aqueous buffers for many compounds of low solubility,often unattainable by other methods. The pKa measurements of compounds withextremely low solubility using multiplexed CE with methanol/water cosolvent buffersare presented. � 2008 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci

Keywords: pKa measurements; multip
lexed capillary electrophoresis; low solubilitycompounds; mixed cosolvent buffers; aqueous buffers; dissociation constants of drugmolecules
INTRODUCTION

The acid–base dissociation constant of substances(pKa value) is a very important parameter in drugdesign and optimization. The degree of ionizationstrongly affects solubility, permeability, and drug

ontains supplementary material, available atce.wiley.com/jpages/0022-3549/suppmat.alytical Technologies, Inc. (Formerly Combi-h Loop Drive, Suite 4200, Ames, IA 50010.rdo’s present address is Novartis Institutes forarch, 250 Massachusetts Avenue, Cambridge,

ce to: Franco Lombardo (Telephone: 617-871-871-3078.; E-mail: franco.lombardo@novartis.

aceutical Sciences

, 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 ofdistribution in humans (VDss)2 has shown thatthe fraction of compound ionized at pH¼ 7.4,together with the fraction of free drug in plasmaare the largest contributors to the prediction ofVDss, via the fraction unbound in tissues ( fut).

In recent years high throughput drug discoveryoperations have brought to focus the need forthe rapid evaluation of various physicochemicalproperties of newly synthesized compounds usingminute quantities3–6 and significant efforts havebeen devoted to developing techniques adaptedto these challenges. There have been a number ofmethods employed for pKa measurements basedon solubility, potentiometric titration,7–10 spectro-photometry,11–14 HPLC15–17 and, most recently,

JOURNAL OF PHARMACEUTICAL SCIENCES 1

2 SHALAEVA ET AL.

on capillary electrophoresis (CE).18–33 The solu-bility method is of limited accuracy and potentio-metric titrations typically require mg amounts ofpure sample with a throughput of 20–40 min percompound.34 In addition, the sample concentra-tions required for potentiometry often result inprecipitation of poorly soluble compounds, neces-sitating mixed solvent extrapolation methods andfurther impacting sample throughput.

A rapid method for pKa measurement employinga mixed-buffer linear pH gradient and spectro-photometric detection was recently demon-strated.35 The spectral gradient analysis (SGA)technique performs pKa measurements sequen-tially from a 96-well plate format using 10 mMDMSO stock solutions and samples are assayed inabout 4 min. Compounds, however, must possessgood chromophores close enough to the center ofionization for the detection of a spectral shift withchanging pH; otherwise, the pKa values may goundetected. Precipitation of low solubility com-pounds is also a limitation of the method, althoughcosolvents could be employed, but data analysisoften requires user intervention and sampleimpurities, degradants or counter ions possessingsimilar UV characteristics may also potentiallyinterfere with the measurement.

The application of CE for medium to highthroughput pKa measurements in support ofdrug discovery projects has steadily increasedin recent years. The many advantages of CEfor pKa measurement were highlighted in severalrecent reviews34,36–38 including: minimal samplerequirement with extremely low sample consump-tion, the ability to separate impurities and/ordegradants from the target compound, sensitiveon-line UV detection, and automated operation.In addition, precise knowledge of sample concen-tration is not required because only compoundmigration times are required for analysis, and nospecial demands are placed upon the purity ofbuffer solutions.

The effective mobility (meff) of a solute, in afield of voltage V, is easily calculated from thedifference in migration time between the com-pound (ta) and a neutral marker (tm), commonlyDMSO, through Eq. (1):

meff ¼LdLt

V

� �1

ta� 1

tm

� �(1)

and this technique is based on the variation of thesolute mobility with the variation of buffer pH.

In Eq. (1) Ld is the length from the capillaryinlet to the detection window and Lt is the total

JOURNAL OF PHARMACEUTICAL SCIENCES

capillary length in cm, and V is the appliedvoltage. A plot of compound meff as a function of pHyields a sigmoidal curve, from which the inflectionpoint(s) corresponds to the apparent pKa value(s)and thus the mobility is correlated with the pKa.Equations relating the measured meff and pHvalues to the pKa value via nonlinear regressionanalysis have been previously well described inthe literature for up to three ionizable groups,which covers the majority of pharmaceuticalcompounds.22,28 Theoretically, there is no restric-tion on the number of ionization equilibria thatcan be considered.34

In Pharmaceutical Discovery operations it isoften of interest to discriminate differences in pKa

values among structural analogs and it is notunusual to encounter molecules with two or morevery close pKas. For the method to be suitable forthe simultaneous measurement of the variety ofpossible pKa combinations, in a high throughputmode, it is necessary to have buffers spaced withrelatively small increments and also covering aswide a pH range as possible. For example,Ishihama et al.22 demonstrated the successfulmeasurement of up to six pKa values for angio-tensin by CE using 19 different buffers from pH1.8 to 12.0.

One approach to increasing sample throughputinvolves the application of multiplexed capillaryarray electrophoresis with UV absorption detec-tion.39,40 Recently, the use of a 96-capillaryarray for the rapid measurement of pKa valuesfor 96 different compounds (mostly monoproticacids and bases) was demonstrated.33 A measure-ment of 128–168 compounds in an 8 h period wasachieved by simultaneously analyzing eight com-pounds over 12 pH values in a single CE run.

A significant challenge for all of the afore-mentioned pKa measurement techniques includ-ing CE is the precipitation of low aqueoussolubility compounds. Unfortunately, the currenttrend in drug discovery is toward compoundspossessing a relatively high lipophilicity and afairly low aqueous solubility.41 Detection limitsusing UV spectrophotometry are reported to be1–10 mM as long as the compound possesses asuitable pH-sensitive chromophore,5 while CEmethods employing low UV wavelength detectionallow measurements at similar concentrationswithout restrictions regarding the position of thechromophore.22

A potential approach for increasing the detec-tion sensitivity and expanding the scope ofdetection involves the integration of mass spectro-

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MULTIPLEXED CE pKa DETERMINATION 3

metry with CE (CE-MS).42,43 Wan et al.43 mea-sured the pKa values for 60 different compoundsincluding some that were sparingly soluble or oflow UV absorbance by CE-MS, and samplethroughput was increased by pooling up to 56compounds. Although promising, the day-to-dayreliability and reproducibility of the methodremains to be assessed and commercially avail-able software for performing mass deconvolutionand data processing would need to be developed toenable routine pKa measurement.

A common approach for the pKa measurementof aqueous insoluble compounds involves the useof different percentages of mixed cosolvent buffersand extrapolation to 0% cosolvent. Typically, atleast three and up to six different percentages ofcosolvent are recommended. Methanol is the mostcommonly employed cosolvent and is consideredto yield the least deviation from a completelyaqueous environment as its effects on pKa havebeen extensively studied.9,44,45

When working in mixed methanol/water solu-tions two different pH scales are commonly used,depending upon how the pH meter is calibratedprior to pH measurement. The notation describingthe different pH scales used within this work isthat recommended by IUPAC for pH quantities46

and that employed by others.45,47–49 The lower-case left-hand superscript indicates the solvent(w for water or s for mixed solvent) in which themeasurement is made, while the lower-case left-hand subscript indicates the solvent where theionic activity coefficient is referred to unity atinfinite dilution (i.e., how the meter was cali-brated). Therefore, the measurement of pH valuesfollowing calibration of the pH meter withstandard buffers prepared, in the same metha-nol/water composition as the sample, yields s

spHvalues. If the pH meter is calibrated with aqueousstandard buffers, measurements in the mixedmethanol/water solutions are in the intersolventalscale and referred to as s

wpH values. It has beenrigorously demonstrated in the literature that thetwo pH scales are related by means of Eq. (2):

sspH ¼ s

wpH þ d (2)

where d is a constant that is dependent strictlyupon the composition of the mixed solvent.47

Eq. (2) assumes that the liquid junction potentialof the potentiometric system used for pH mea-surement is negligible, as was previously demon-strated for the same type of combination glasselectrode used in this work.47 Values for d have

DOI 10.1002/jps

been previously determined over a wide range ofmethanol/water compositions.48

Measurements of meff as a function of swpH value

using CE yield a compound’s apparent swpKa

value(s) for that particular solvent compositionand ionic strength. The s

wpKa value can beconverted to a s

spKavalue via Eq. (3):

sspKa ¼ s

wpKa þ d (3)

Alternatively, the sspKa value can be obtained

directly for a compound working in the sspH scale

by employing CE with cosolvent buffers prepar-ed from equimolar mixtures of an acid and itscorresponding salt50–52 or by using pH metercalibration standards with known s

spKa values(e.g., potassium hydrogenphthalate).53,54 How-ever, use of the s

wpH scale combined withEqs. (2) and (3) provides a much more practicalapproach when working with mixed solventsystems.

The most popular extrapolation method is theYasuda–Shedlovsky (Y–S) method, which relatessspKa to the inverse of the dielectric constant of thebinary solvent (1/e) by Eq. (4):

sspKa þ log½H2O� ¼ a

"þ b (4)

where a and b are constants. Extrapolation toe¼ 78.3 and log H2O¼ 55.5 (the dielectric constantand molar concentration of pure water, respec-tively) yields the apparent w

wpKa value. Previousstudies have demonstrated that Y–S extrapola-tion generally yields a linear relationship andaccurate w

wpKavalues when using solvent mixturespossessing e> 50, which for methanol correspondsto <60 wt% or <65.5% (v/v).5,44

Although numerous publications have appear-ed describing the application of CE for performingaqueous pKa measurements, relatively few haveexplored the use of methanol/water cosolvents.Bellini et al.55 described the measurement of pKa

values in increasing methanol concentrations andextrapolation to 0% cosolvent on a set of five acids.Due to the limited throughput (up to 40 min perseparation) the ‘‘two pH points per compound atfour different methanol concentrations’’ approachwas recommended as more practical, requiringonly eight runs per compound. Buckenmaieret al.45 measured by CE the s

wpKa values ofseveral basic compounds commonly used as testprobes in HPLC over varying methanol contents.A few other studies have measured the s

spKavaluesfor compounds using CE in pure methanol.50–52


4 SHALAEVA ET AL.

In the present work we report and discussthe measurement of compound pKa values withmultiplexed 96-capillary array electrophoresisemploying fixed wavelength UV detection at214 nm and 24 pH point aqueous or methanolcosolvent buffers. The goals of this study wereto validate the method employing a diverse setof acidic, basic, and multiprotic compounds; toevaluate issues related to the number of pHbuffers used and its relation to the accuracy andthe resolution of closely spaced pKa values;to assess the compound solubility limits foraqueous pKa measurement; and to investigatethe use of cosolvent extrapolation methods forthe measurement of insoluble compounds bymultiplexed CE. As a test set we used almostexclusively drug compounds spanning a widerange of structures, physico-chemical propertiesand, importantly, containing many examples ofmultiprotic compounds.

EXPERIMENTAL

Apparatus

Two different multiplexed 96-capillary arrayelectrophoresis systems (Advanced AnalyticalTechnologies, Inc., Ames, IA) were utilized in thisstudy: a MCE 2000 (Pfizer Laboratory, Groton,CT) and a cePRO 9600TM (Advanced AnalyticalLaboratory), the first and second generationsystems, respectively, employing slightly differ-ent configurations and experimental methods asdiscussed in the procedures below. Only a briefdescription of the instrument is given here; a morecomplete discussion can be found in earlierreports.33,39,40

pH measurements were taken with a Ross8102 combination glass electrode, equipped withan automatic temperature compensation (ATC)probe, using a Thermo Orion 520Aplus pH meter.The relative pH accuracy of the meter was specifi-ed to be �0.002 pH units.

Chemicals

The test set compounds were ordered from variouscommercial 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 reagentswere purchased from Sigma–Aldrich and used asreceived.


Aqueous and methanol/water mixed cosolventpH buffer kits along with a common 10� outletreservoir buffer concentrate (100 mM sodiumtetraborate) were prepared by Advanced Analy-tical. The 10� outlet buffer concentrate wasdiluted with doubly distilled H2O prior to use.The sets of 24 aqueous pH buffers were preparedfrom phosphoric acid, formic acid, sodium acetate,sodium phosphate, or boric acid with the additionof sodium chloride or/and sodium hydroxide invarious proportions to a level ionic strength ofI¼ 50 mM. The sets of mixed cosolvent buffers(also I¼ 50 mM) were prepared from similarinorganic buffers with the addition of 30%, 40%,50%, or 60% (v/v) methanol. The pH values of themixed cosolvent buffers were measured aftermixing with methanol using a pH meter cali-brated with standard aqueous buffers. Themeasured values are therefore described as s

wpHvalues (see Introduction Section).17,47,48 The setsof aqueous and mixed cosolvent buffers werestored in the lab tightly closed in the plasticbottles and used for a period of 3–5 months in mostcases. The pH values of the buffers were generallystable to �0.04 pH units when stored tightlysealed between uses. To ensure the highest level ofaccuracy over long term storage, particularly inthe buffers above pH 9.0, it is recommended toperiodically measure the pH values using acalibrated pH meter.

Sample Preparation

Samples were prepared by weighing 0.2–1.0 mg ofcompound followed by addition of solvent to yield aconcentration of 50–150 mg/mL (ppm). In the caseof some low solubility compounds, samples couldbe further diluted to approximately 5–10 mg/mL toavoid precipitation in aqueous buffer experi-ments. The solvent consisted of 0.1–0.2% (v/v)DMSO (neutral marker) and 1–5 mM HCl orNaOH to enhance the solubility of basic or acidiccompounds, respectively. Some low solubilitycompounds were initially solubilized in 60% (v/v)methanol prior to addition of aqueous solution.A few compounds (e.g., indomethacin) wereobserved to appreciably degrade following pre-paration in HCl or NaOH containing solvents (seeResults and Discussion Section). Sample solutionswere sonicated for 30 s and filtered through a0.20 mm nylon syringe filter if necessary. Whenperforming mixed cosolvent experiments it wasbeneficial to prepare samples at the lowest

DOI 10.1002/jps


possible concentration and in the presence of 60%methanol (v/v) to minimize effects from compoundprecipitation and improve the peak shape of theDMSO marker peak during the separation.

Procedures

Prepared sample solutions were dispensed into a300 mL Costar 96-well microplate (300 mL/well) orinto a 200 mL skirted PCR plate (50 mL/well)depending on the instrument used. A 24 point pHexperiment required 24 wells to be filled with eachsample and, with this protocol, four compoundscould be analyzed simultaneously.

A deep well (1.0 mL working volume) 96-wellmicroplate was employed as the pH buffer platefor analysis. The buffer plate was prepared freshfor each experiment. Each of the 24 pH buffers(1.1 mL/well) was dispensed consecutively in wellsA1–B12 for the first sample, wells C1–D12 for thesecond sample and so forth.

The buffer plates utilized in the mixed cosolventexperiments were prepared in a similar layout tothe aqueous buffer plates. Each cosolvent bufferplate was prepared with a common percentageof methanol (v/v): 60%, 50%, 40%, or 30%. Thesample plate was sequentially analyzed with eachcosolvent buffer plate, from 60% to 30% methanolcontent, to acquire the corresponding s

wpKa

values.

pKa Measurements

Experiments performed with the MCE 2000system used a 96-capillary array of bare fusedsilica capillaries (75 mm i.d., 150 mm o.d.) witheffective and total lengths of 55 and 80 cm,respectively. Prior to each experiment the capil-laries were flushed with water followed by thesodium tetraborate outlet buffer (pH¼ 9.0) for5 min each at 40 psi. Following the capillary flush,the pH buffer plate was loaded and the capillarieswere filled with the different pH buffers for 8 minat �2.0 psi vacuum. The sample plate was nextinserted into the instrument and the sampleswere injected at �1.0 psi for 5 s. The buffer platewas placed back into the instrument immediatelyafter sample injection and the electrophoresisseparation started. Aqueous pKa measurementswere performed at þ6 kV for 18 min, whilecosolvent measurements were run at þ5 kV for25 min, with �0.5 psi vacuum level applied duringCE in both cases.

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Experiments performed on the cePRO 9600TM

system utilized slightly different operating con-ditions. The 96-capillary array of bare fused silicacapillaries (75 mm i.d., 150 mm o.d.) had effectiveand total lengths of 33 and 55 cm, respectively.The capillary array was flushed with waterfollowed by the sodium tetraborate outlet buffer(pH¼ 9.0) each morning for 3 min at 50 psi. At thestart of an experiment the pH inlet buffer plate, anempty 1.0 mL 96-well waste plate and up to two96-well sample plates are loaded onto the stage.The capillaries are initially flushed with outletbuffer for 1 min at 50 psi and then the capillariesare filled with the different pH buffers for 2 minat �2.0 psi vacuum and, subsequently, samplesare injected at �0.1 psi for 20 s. The inlet pHbuffer plate is returned into position and theseparation is started. For aqueous experimentsthe separation was performed at þ3.5 kV for13 min with a �0.2 psi vacuum applied during CE.For cosolvent experiments, the samples wereinjected at �0.1 psi for 50 s and separated atþ3.5 kV for 18 min with the addition of �0.25 psivacuum. No flushing of the capillaries wasrequired between runs with the exception of the1 min outlet buffer preflush at the beginning ofeach analysis cycle.

pKa Data Analysis

Versions 1.01–4.03 of the program pKa Estima-torTM (Advanced Analytical Technologies, Inc.)were used to calculate the pKa values of com-pounds from the electrophoresis data. The separa-tion data were imported into the program wherethe effective mobility (meff) was calculated for eachpH value from the difference in migration times ofthe compound (ta) and DMSO marker (tm) usingEq. 1. The meff was then plotted as a function of pHto yield a sigmoidally-shaped titration curve. Themolecular weight (MW) of the compound wasentered into the software and the compoundcharge (Zc) was estimated by the software usinga previously described empirical relationshipbetween MW, maximum meff and Zc.

28 From thecalculated compound charge, a suitable equationfrom a set of nine different model equationsrepresenting up to three ionizable groups28,22 wasselected by the software to perform a nonlinearregression analysis on the data. When performinga cosolvent experiment, the final result is theapparent s

wpKa (I¼ 50 mM) at the particular %methanol concentration. Each s

wpKa result is


6 SHALAEVA ET AL.

converted to its respective sspKa value by use of the

appropriate d term.48 The apparent sspKa values

are then plotted as a function of the reciprocal ofthe solvent dielectric constant (1/e) (Y–S method)9,48,56,57 to 0% cosolvent to yield the apparent w

wpKa

value (I¼ 50 mM) according to Eq. (4).

RESULTS AND DISCUSSION

1. Aqueous pKa Measurements

Comparison to Literature Values

The test set compiled for the multiplexed CEmethod validation included a total of 105 com-pounds (166 total pKa values) of which most werecommercially available drugs. Table 1 lists theresults for compounds that were successfullymeasured with aqueous buffers (98 of 105). Anadditional seven compounds were found to pre-cipitate during aqueous analysis and their pKa

values were measured using methanol cosolventbuffers (see Experimental Section). We attemptedto make the validation set as diverse as possible tothoroughly evaluate various combinations ofphysicochemical properties encountered withina Pharmaceutical Discovery setting. Nearly half(49 of 105) of the compounds in the validation setare multiprotic, having from two to five pKa

values. Included are basic, acidic, and zwitterioniccompounds.

The range of pKa values measurable by multi-plexed CE employing the 24-point aqueous bufferswas approximately equal to the pH range of thebuffer series (pH 1.75–11.20). Accordingly, wewere able to measure several pKa values below 2.0(buspirone pKa¼ 1.93; nicotinic acid pKa¼ 1.86;piroxicam pKa¼ 1.81; sulfacetamide pKa¼ 1.26)and above 11.0 (cefuroxime pKa¼ 11.30; sulfa-salazine pKa¼ 11.31). We were unable to repro-ducibly 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 withprevious studies which found that it was possibleto fit pKa values from data encompassing only aportion of the titration curve.20,22

All of the compounds in the test set werewell detected at concentrations of 50–100 ppm(mg/mL). However, several compounds (identifiedby superscript a) were observed to precipitate atthe typical working concentration when thepH value approached that corresponding to theneutral form of the compound. The sensitivity


provided by low UV detection at 214 nm allowedfor dilution of these insoluble compounds up to10-fold and for successful measurement of theirpKa values in aqueous buffers without precipita-tion. Methanol cosolvent buffers were employed toanalyze compounds that were still observed toprecipitate at concentrations approaching theirdetection limits.

Compounds were typically dissolved in 1 mMHCl (for bases) or 1 mM NaOH (for acids) toimprove their solubility during sample prepara-tion. However, a few compounds were observed topartially degrade during the time course of theanalysis when dissolved in 1 mM acid or base(identified by superscript b). As it could be assumedthat the intact compound would possess thehighest MW relative to any degradation productsand therefore the lowestmeff it was possible in mostcases to measure the pKa value of the intactcompound in the presence of degradant impurities.Alternatively, preparation of unstable compoundsin water was also found to minimize degradation.

For the majority of compounds in the test setmultiple literature pKa values have been reportedusing a variety of experimental methods andconditions. We felt it was important and mostappropriate to include all verifiable results in ourcomparisons as opposed to citing results from asingle source or method. This approach allowsfor a more complete assessment of our results incomparison to previous studies.

The pKa values measured in this work arereported as apparent pKa values at I¼ 50 mM. Animportant factor to note when comparing experi-mental data is that the pKa literature values arereported over a typical range of ionic strengthsfrom I¼ 0 mM (thermodynamic) to 150 mM. Whenconverting from an ionic strength of 150 to 0 mMthe correction factor at 258C would be þ0.12 for amonoacid and �0.12 for a monobase. A majorityof potentiometric and spectroscopic data arereported as apparent pKa values at I¼ 150 mM.The correction between pKa values at I¼ 50 mMmeasured in this work and I¼ 150 mM would be�0.04 for monoacids or monobases, �0.11 for theweaker pKa (z¼ 2) of diacids or dibases, and �0.18for the weakest pKa (z¼ 3) of triacids or tribases. Asignificant amount of literature results have beenconverted to I¼ 0 mM, especially for CE-basedmeasurements. For the weaker pKa of a diacid ordibase, conversion from I¼ 150 mM to I¼ 0 mMrepresents a span of �0.36 pKa units. Somereferences either do not mention the ionicstrength or do not clearly state whether or not a

DOI 10.1002/jps

Table 1. Summary of pKa Values Measured by 96-Capillary Array CE Using Aqueous Buffers and a Comparison toLiterature Values

CompoundpKa Average,

This WorkpKa Average,

Literature Difference pKa Range References

1 Abacavir 5.04 5.01 0.03 5.01 662 Acebutolol 9.52 9.48 0.04 9.37–9.56 27,33,35,67,683 Acetaminophen

(paracetamol)9.41 9.56 �0.15 9.45–9.75 14,27,33,35,43,59,69

4 Acetylsalicylic acid 3.49 3.50 �0.01 3.30–3.74 27,28,30,35,695 Acyclovir 2.20 2.23 �0.03 2.16–2.34 5,28,35,70

9.18 9.22 �0.04 9.04–9.316 Alprenolol 9.62 9.52 0.10 9.38–9.59 27,30,35,687 4-Aminobenzoic acid 2.17 2.46 �0.29 2.45–2.46 5,12

4.76 4.81 �0.05 4.62–4.998 Aminopyridine, 2- 6.71 6.72 �0.01 6.70–6.76 21,28,349 Aminopyridine, 4- 9.22 9.19 0.03 9.02–9.29 21,26,28,3410 Amitriptylinea 9.51 9.41 0.10 9.32–9.49 5,45,51,71,7211 Atenolol 9.60 9.54 0.06 9.42–9.64 26,27,28,29,30,33,35,43,67,69,73,7412 Betahistine 3.90 4.26 �0.35 3.46–5.21 27,75,76

10.02 9.98 0.04 9.78–10.1313 Bifonazole 6.29 5.8 0.49 5.72–5.88 77 c

14 Buspirone 1.93 67,697.64 7.6 0.04 7.60

15 Cefadroxil 2.55 2.66 �0.11 2.47–2.86 23,287.21 7.38 �0.17 7.14–7.599.71 9.89 �0.19 9.89

16 Cefuroximeb 2.14 2.11 0.02 2.04–2.17 2311.30

17 Cephalexinb 2.61 2.66 �0.05 2.34–3.11 23,27,35,597.08 7.02 0.06 6.79–7.14

18 Cetirizine 2.24 2.11 0.13 2.10–2.12 78,793.47 2.91 0.56 2.90–2.937.90 7.99 �0.09 7.98–8.00

19 Chloroamphetamine 9.85 9.80 0.05 9.80 8020 Chloroquine 7.44 8.25 �0.81 8.10–8.50 81,82,83,84

10.68 10.37 0.31 9.94–10.8721 Chlorthalidone 8.98 9.11 �0.13 9.11 35

10.82 10.98 �0.16 10.9822 Cimetidine 6.90 6.82 0.08 6.68–6.97 27,28,33,4323 Clomipraminea 9.57 9.28 0.29 9.17–9.38 71 c

24 Clotrimazolea 5.99 5.89 0.10 5.48–6.30 85 c

25 Clozapine 4.02 4.08 �0.06 3.58–4.40 5,43 c

7.60 7.82 �0.22 7.63–7.9426 Codeine 8.15 8.11 0.04 7.81–8.22 5,29,33,43,45,6927 Deprenyl 7.47 7.43 0.04 7.40–7.48 5,68,6928 Desipramine 10.42 10.27 0.15 10.16–10.65 5,35,51,72,74,8629 Dichlorphenamide 8.24 8.3 �0.06 8.20–8.41 28,87 c

9.50 10 �0.50 9.82–10.2730 Diphenhydramine 9.23 9.13 0.10 9.10–9.16 5,4531 Emetine 6.37 7.02 �0.65 6.68–7.36 28,88

8.81 8.57 0.23 8.23–8.9132 Eserine

(physostigmine)8.15 8.15 0.00 8.13–8.17 14,35

33 Flufenamic acida 3.97 4.02 �0.05 3.63–4.27 5,27,33,6734 Flumequine 6.31 6.36 �0.05 6.09–6.65 5,14,26,28,33,35,43,54,59,69

(Continued)

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Table 1. (Continued )




35 Furosemide 3.60 3.55 0.05 3.34–3.74 14,27,28,33,35,43,70,74,8910.35 10.46 �0.11 10.15–10.90

36 Histamine 5.80 6.04 �0.25 6.04 35,909.96

37 Hydroquinine 4.32 4.29 0.03 4.29 829.06 9.01 0.05 9.01

38 Ibuprofen 4.35 4.34 0.01 4.01–4.51 14,27,28,29,34,35,43,59,70,89,91,9239 Imipraminea 9.58 9.5 0.08 9.34–9.66 5,34,35,43,49,51,72 c

40 Indomethacinb 4.18 4.29 �0.11 4.06–4.51 5,27,33,35,43,66,9341 Ketoconazole 3.18 3.15 0.03 2.90–3.29 28,35,43,94

6.30 6.39 �0.09 6.22–6.5142 Labetalol 7.35 7.46 �0.11 7.42–7.48 14,35,89,95

9.16 9.38 �0.22 9.32–9.4243 Lamivudine 4.24 4.3 �0.06 4.30 9644 Lansoprazole 4.22 3.99 0.23 3.82–4.15 30,97,98

8.58 8.79 �0.21 8.73–8.8445 Leucovorin

(folinic acid)<1.5 5,99

3.59 3.10 0.49 3.105.12 4.68 0.44 4.56–4.80

10.39 10.28 0.11 10.15–10.4046 Levallorphan 9.26 9.29 �0.03 8.81–9.77 60 cd

10.7447 Lidocaine 7.96 7.91 0.05 7.83–7.98 29,33,35,43,86,91,9248 Maprotiline 10.57 10.20 0.37 10.20 4949 Mebendazole 3.27 3.43 �0.16 3.43 43

9.56 9.93 �0.37 9.9350 Methotrexate

(amethopterin)3.30 3.24 0.06 3.04–3.37 5,28,43

3.86 3.9 �0.04 3.80–4.005.31 5.23 0.08 4.99–5.39

51 Metoprolol 9.60 9.51 0.09 9.36–9.60 27,30,33,35,73,7452 Metronidazole 2.49 2.38 0.11 2.38 3353 Morphine 8.07 8.19 �0.12 8.17–8.21 59,69,91

9.20 9.3 �0.10 9.26–9.3354 Nafronyl 9.0655 Naloxone 8.09 7.94 0.15 7.94 60

9.03 9.44 �0.41 9.4456 Naproxen 4.27 4.13 0.14 4.01–4.20 27,33,74,8957 Nefazodonea 6.7658 Nefopam 8.34 8.98 �0.64 8.65–9.31 10059 Nicotine 3.28 3.14 0.14 3.00–3.25 21,26,28,29,34,43

8.20 8.14 0.06 8.02–8.2960 Nicotinic acid 1.86 2.18 �0.33 2.00–2.43 14,29,30,33,35,43,101

4.71 4.74 �0.03 4.62–4.8461 Nifedipine 2.17 2.60 �0.43 2.60 2862 Norephedrine 9.1263 Norfloxacin 6.14 6.23 �0.09 5.94–6.34 5,28,35,54,66,102,103,104

8.31 8.49 �0.18 8.22–8.7564 Nortriptyline 10.29 10.11 0.18 10.02–10.19 5,45,49,5165 Omeprazole 4.25 4.13 0.12 3.94–4.40 30,43,97,105,106

JOURNAL OF PHARMACEUTICAL SCIENCES DOI 10.1002/jps

8 SHALAEVA ET AL.





8.66 8.86 �0.21 8.70–8.9866 Pantoprazole 3.83 3.77 0.06 3.56–3.92 30,97,106

8.04 8.19 �0.15 8.18–8.1967 Papaverine 6.33 6.34 �0.01 6.21–6.49 14,26,29,33,35,43,59,67,10768 Penicillin Gb 2.64 2.75 �0.11 2.75 10869 Perfenazine 3.64 3.59 0.04 3.59 109

7.76 7.82 �0.06 7.8270 Phenylalanine 2.16 2.19 �0.03 2.18–2.20 91,110

9.01 9.08 �0.07 9.0871 Pindolol 9.49 9.64 �0.15 9.54–9.74 33,10772 Piroxicam 1.81 2.25 �0.45 1.88–2.53 5,27,33,35,74,103

5.28 5.18 0.10 4.94–5.3273 Prazosin 7.12 7.08 0.04 7.04–7.11 5,3574 Procaineb 2.14 2.28 �0.14 2.27–2.29 33,35,67,69,92

9.05 9.06 �0.01 9.01–9.1575 Promazinea 9.56 9.11 0.45 8.92–9.40 72,10976 Promethazinea 9.05 8.93 0.12 8.62–9.10 5,68,72,10977 Propranolol 9.59 9.51 0.08 9.14–9.61 27,28,29,30,35,43,67,68,70,74,92,

10778 Pyrilamine 4.14 3.99 0.15 3.99 34,67

9.12 9.14 �0.02 9.10–9.1879 Quinine 4.29 4.21 0.08 3.95–4.46 13,14,21,26,28,29,33,35,43,45,59,

68,82,918.53 8.51 0.02 8.35–8.60

80 Salicylic acid 2.85 2.90 �0.05 2.64–3.08 21,26,27,28,30,33,34,35,43,59,6981 Sotalol 8.38 8.29 0.09 8.25–8.35 35,107,111

9.47 9.82 �0.36 9.72–9.9882 Sulfacetamide 1.26 1.85 �0.59 1.76–1.95 14,35,43

5.32 5.23 0.09 5.16–5.3083 Sulfasalazine 2.27 2.53 �0.27 2.40–2.65 5,112

7.87 7.95 �0.08 7.91–9.7011.31 11.2 0.11 10.51–11.8

84 Sulpiride 9.04 9 0.04 8.95–9.13 27,33,35,66,113,11410.10 10.02 0.08 9.79–10.19

85 Terbutaline 8.79 8.58 0.21 8.23–8.72 13,14,27,35,43,74,1079.54 9.95 �0.41 9.89–10.00

10.47 10.89 �0.43 10.58–11.1086 Tetracaine 2.29 2.3 �0.01 2.20–2.39 35,66,92

8.50 8.44 0.06 8.29–8.5587 Tetracycline 3.36 3.31 0.05 3.30–3.33 35,115,116

7.09 7.51 �0.42 7.16–7.709.17 9.54 �0.37 9.43–9.69

88 Theophylline 8.51 8.62 �0.11 8.56–8.66 27,35,9189 Thiopropazate 3.07 3.2 �0.13 3.20 117

7.66 7.15 0.51 7.1590 Trazodone 6.80 6.75 0.05 6.69–6.79 33,49 c

91 Trimethoprim 6.88 6.84 0.04 6.6–7.07 66 c

92 Trimipraminea 9.56 9.25 0.31 9.15–9.37 49 cd

93 Tripelennamine 4.08 4.20 �0.12 4.20 869.10 8.71 0.39 8.71

94 Tryptophan 2.30 2.38 �0.08 2.30–2.60 22,26,27,28,29,43

(Continued)

DOI 10.1002/jps JOURNAL OF PHARMACEUTICAL SCIENCES






9.22 9.35 �0.14 8.97–9.4395 Tyrosine 2.18 2.19 �0.01 2.18–2.2 22,35,66,91,118

8.76 9.12 �0.36 8.94–9.2110.03 10.21 �0.18 9.99–10.47

96 Vancomycin 2.78 2.66 0.12 2.66 1076.96 7.49 �0.53 7.498.37 8.63 �0.26 8.638.76 9.26 �0.50 9.26

10.06 10.16 �0.10 10.1697 Verapamila 8.93 8.81 0.12 8.66–9.07 5,22,27,28,35,58,74,10998 Warfarin 4.97 4.94 0.03 4.70–5.15 5,22,26,27,28,29,33,35,43,92 c

aAnalyzed at 10–50 ppm to reduce compound precipitation.bSome compound degradation was observed when prepared in 1 mM acid or base.cPotentiometric measurement performed by pION, Inc. (Woburn, MA).dPotentiometric measurement Pfizer, Sandwich Laboratories, Sandwich (UK).

10 SHALAEVA ET AL.

correction was applied. Also, the correction forzwitterionic compounds is not straightforwardand often authors report apparent pKa values forzwitterions while correcting acidic and basiccompounds to I¼ 0 mM.

Another source of data variability stems fromthe temperature during the experiment. For ourmeasurements, room temperature air was circulat-ed around the capillary array by cooling fans inthe instrument during the CE separation. In addi-tion, relatively low field strengths (60–80 V/cm)were employed to reduce the effects of Jouleheating and maintain a constant temperature ofapproximately 20–258C. Indeed, the current levelreached a steady state within 1–2 min and did notsignificantly increase throughout the CE separa-tion. In some cases the temperature effect may besignificant, as when Hasegawa et al.58 reporteda shift of 0.33 pKa units for verapamil whenincreasing from 20 to 378C. Nevertheless, tem-perature is often omitted in the literature dataand we used pKa values as found or within therange of 20–258C if there was a choice.

The medium employed when performing pKa

measurements (i.e., aqueous or cosolvent) mayalso slightly influence results relative to others.Previously reported pKa values for many spar-ingly soluble compounds listed in Table 1 wereobtained with potentiometry using Y–S cosolventextrapolation methods. It has been previouslyshown that the Y–S method slightly undere-stimates pKa values for basic compounds andoverestimates pKa values for acidic compoundswhen compared to aqueous-based measurements(see Sample Preparation Section).59


The variability in experimental methods, condi-tions, and data presentation described aboveinevitably brings additional challenges when com-paring results. For this reason, we decided topresent not only the average of pKa values found inthe literature, but also present the range of reportedvalues as seen in Table 1. We found that the averagerange in literature values for the compounds listedin Table 1 is approximately 0.35 pKa units, with theresults for some compounds varying by greater than0.9 pKa units (e.g., betahistine, chloroquine, leval-lorphan, and sulfasalazine).

The overall average difference between thepKa values measured in this work to the averageliterature pKa values was found to be �0.04 Uwhile the absolute average error was 0.16, indi-cating a very good level of agreement to previouslyreported results when considering the variablesdiscussed above.

Figure 1 shows the correlation between the pKa

values measured in this work to the average ofavailable literature pKa values. Eight pKa valueswere excluded from the correlation as we wereunable to ultimately confirm or identify a suitableliterature reference. For example, we found onlyone pKa value reported for buspirone, cefuroximeand histamine while we observed two pKa valuesby our measurement. Kaufman et al.60 predictedtwo pKa values for levallorphan, but they wereable to measure only one due to a decreasingsolubility of the compound. We were unable toidentify any reliable literature pKa values fornafronyl and norephedrine.

The correlation of Figure 1 yielded a slope of0.997 with an intercept of þ0.059 (R2¼ 0.993)

DOI 10.1002/jps

Figure 1. Correlation of aqueous pKa values measured by multiplexed 96-capillaryarray CE with the average of pKa values reported in the literature.


providing further validation of the multiplexedCE method for pKa measurement. It should benoted that the correlation is somewhat affected bythe results obtained at the pH extremes, wheremeasurements are inherently more difficult byany method due to the presence of significantamounts of Hþ or OH� species. For example, thelower pKa values reported for sulfacetamide in theliterature were 1.76, 1.95, and <2.5 as comparedto our average result of 1.26. This data point,being the lowest in the set, does have some effecton the correlation. However, its removal yieldsa slope of 1.000 with an intercept of þ0.037(R2¼ 0.993) and thus the variation is of littlepractical significance.

Interlaboratory Comparisons of pKa Results

To evaluate the interlaboratory reproducibility ofthe multiplexed CE method for pKa measurementwe examined compounds within the test set whichwere analyzed in both laboratories (Tab. 2, n¼ 30compounds, 53 pKa values). Good overall agree-ment was obtained between the two laboratoriesconsidering the slight differences in instrumentdesign, capillary length, and experimental para-meters. The average difference between measur-ed pKa values was �0.02 U (0.12 average absoluteerror) 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 wereacquired over several months or longer with atleast two different lots of aqueous buffers, asopposed to repetitive measurements on the sameday, for which the SD is most often 0.05 U or less.

Upon closer examination of Tables 1 and 2 it canbe observed that often the largest discrepanciesbetween results occur for multiprotic compoundswith closely spaced pKa values (less than 2 pHunits apart). In particular, several compoundsvaried considerably between laboratories and/orpossessed relatively high standard deviations,namely chloroquine, indomethacin, levallorphan,methotrexate, and tetracycline. We sought toexplore these differences and further investigatethe multiplexed CE method in several areas—inregard to the resolution of closely spaced pKa

values, the ability to identify potential compounddegradation or impurities, and the lower limitof compound solubility for performing aqueousmeasurements. We then describe a method forthe pKa measurement of aqueous insoluble com-pounds using mixed methanol/water cosolventbuffers and extrapolation to 0% cosolvent.

Resolution of Closely Spaced pKa Values

The measurement of closely spaced pKa valuesless than 2 pH units apart requires carefulattention to experimental parameters along with


Table 2. Interlaboratory Comparison of pKa Values Measured by 96-Capillary Array CE Using Aqueous Buffers(See Experimental Section for Differences Between Methods)

CompoundAverage pKa

cePRO SDAverage

pKa MCE 2000 SDDifference

cePRO–MCE2000

1 Acyclovir 2.20 0.03 2.20 0.07 0.009.20 0.01 9.17 0.01 0.03

2 Betahistine 3.88 0.02 3.93 0.11 �0.059.98 0.06 10.07 0.14 �0.09

3 Chloroquine 7.27 0.09 7.77 0.10 �0.5010.64 0.07 10.75 0.03 �0.11

4 Cimetidine 6.93 0.06 6.85 0.01 0.085 Codeine 8.14 0.06 8.18 0.15 �0.046 Flumequine 6.29 0.05 6.34 0.06 �0.057 Furosemide 3.60 0.06 3.59 0.05 0.01

10.39 0.05 10.31 0.04 0.088 Ibuprofen 4.34 0.06 4.37 0.07 �0.039 Indomethacin 4.01 0.09 4.36 0.31 �0.3510 Ketoconazole 3.13 0.07 3.25 0.19 �0.12

6.35 0.05 6.25 0.12 0.1011 Labetalol 7.35 0.03 7.36 0.05 �0.01

9.17 0.04 9.14 0.19 0.0312 Levallorphan 9.22 0.02 9.29 0.05 �0.06

10.57 0.15 10.86 0.29 �0.2913 Lidocaine 7.97 0.06 7.95 0.03 0.0214 Methotrexate (amethopterin) 3.27 0.07 3.36 0.08 �0.09

3.53 0.11 4.30 0.38 �0.775.15 0.04 5.51 0.15 �0.36

15 Morphine 8.13 0.05 7.99 0.13 0.149.31 0.06 9.07 0.04 0.24

16 Naloxone 8.01 0.04 8.15 0.09 �0.149.13 0.03 8.96 0.21 0.17

17 Norfloxacin 6.18 0.04 6.12 0.06 0.068.37 0.05 8.26 0.01 0.11

18 Papaverine 6.38 0.05 6.27 0.06 0.1119 Piroxicam 1.87 0.06 1.74 0.16 0.13

5.34 0.06 5.21 0.04 0.1320 Propranolol 9.59 0.05 9.58 0.16 0.0121 Quinine 4.33 0.07 4.24 0.07 0.09

8.49 0.07 8.57 0.05 �0.0822 Sotalol 8.31 0.03 8.45 0.05 �0.14

9.59 0.01 9.35 0.08 0.2423 Sulpiride 9.01 0.03 9.08 0.07 �0.07

10.09 0.05 10.11 0.05 �0.0224 Terbutaline 8.79 0.02 8.79 0.10 0.00

9.60 0.02 9.46 0.28 0.1425 Tetracaine 2.25 0.05 2.34 0.10 �0.09

8.51 0.04 8.49 0.08 0.0226 Tetracycline 3.36 0.09 3.36 0.05 0.00

7.09 0.88 7.10 0.69 �0.019.30 0.23 9.00 0.48 0.30

27 Trimipramine 9.51 0.02 9.61 0.16 �0.1028 Tripelennamine 4.07 0.05 4.10 0.07 �0.03

9.09 0.00 9.10 0.04 �0.0139 Tyrosine 2.23 0.02 2.11 0.04 0.13

8.85 0.06 8.69 0.09 0.1610.06 0.06 10.01 0.14 0.05

30 Warfarin 4.94 0.03 5.02 0.14 �0.08

JOURNAL OF PHARMACEUTICAL SCIENCES DOI 10.1002/jps

12 SHALAEVA ET AL.


data analysis and interpretation. For potentio-metry, the sample should be fully solubilized andof known concentration to accurately determinethe number of bound protons per molecule ofsample and to identify closely spaced pKa valuesby means of a Bjerrum plot.5 In addition, the finiteelectrode response time results in relatively longanalysis times when sampling at many differentpH points during the titration. When employingUV spectrophotometric methods, difficulties arisefrom overlapping spectra less than two pKa unitsapart. In such cases, target factor analysis (TFA)is required to deconvolute the results. It wasrecommended to predict in advance how manypKas are expected and their approximate valuessince at times TFA can fit the same data for two,three, or four pKa values.35

When measuring closely spaced pKa values byCE it is important for the buffers used to be ofknown pH value, evenly spaced, and of equal ionicstrength throughout the pH range. It has beenpreviously suggested that five pH points should beused per one pKa value measured.61 The majorityof previously reported CE methods used 10–12evenly spaced buffers to cover the pH range from 2to 11, translating to a spacing of 0.8–0.9 pH units.This spacing can impact the accurate measure-ment of pKa values closer than approximately3 pH units apart. Increasing the number of buffersis desirable when analyzing compounds withmultiple pKa values as shown by Ishihamaet al.,22 who measured up to six or seven ionizablegroups employing CE with 19 evenly spacedbuffers from pH 2 to 12.

The increase in throughput afforded by multi-plexed CE in combination with the above con-siderations led us to develop a 24 point buffersystem covering the pH range from 1.7 to 11.2,decreasing the spacing to 0.4 pH units. Evenspacing of the buffers was maintained to max-imize the precision of measurements across theentire pH range. In addition, we noticed that thereproducibility suffered if the pH values betweenbatches were not exactly reproduced, especiallywhen measuring multiprotic compounds withthe pKa values less than 3 U apart.

Another factor influencing the measurementof closely spaced pKa values is the separationresolution between the compound and neutralmarker. To improve the separation resolution, thevacuum level applied to the capillary array in thiswork was minimized to yield a separation time of10–13 min, as compared to previous CE studieswhere separation times on the order of 1–5 min

DOI 10.1002/jps

were obtained by the use of high pressures.Separation resolution is particularly importantwhen close pKa values are of different types asin acid/base zwitterions, where the meff changesfrom positive to negative causing inversion ofthe migration order surrounding the compoundpI value. Examples of zwitterionic compoundswith closely spaced pKa values measured in thisstudy include labetalol, methotrexate, morphine,naloxone, sotalol, and sulpiride. From Table 2, theinterlaboratory pKa differences for most multi-protic zwitterions were within 0.2 pKa units. Thelargest discrepancy was for methotrexate, whichhas three pKa values within two pH units of eachother. The differences in results were most likelydue to subtle variations in the buffer pH valuesand/or slight differences in separation resolutioncaused by differences in capillary length andapplied voltage between labs.

Importantly, the use of a previously developedempirical relationship between compound MW,meff and charge to predict the number of pKa

values present is vital when analyzing compoundswith closely spaced and/or unknown pKa values.28

In the case of methotrexate, a predicted charge ofþ0.95 and �1.76 indicated that the compound wasa monobase/diacid with three pKa values. Simi-larly, three pKa values were reported by Milleret al.28 using the aforementioned empiricalrelationship and by Bergstrom et al.62 using UVspectrophotometry. However, only two pKa valueswere reported by Wan et al.43 using CE-MS and10 widely spaced pH buffers. This examplehighlights the need to use meff and MW as anindicator of compound charge when measur-ing pKa values by CE-based methods.

In some cases only partial resolution wasobtained between the compound and neutralmarker at pH points near the pI (isoelectric point)value making the assignment of migration timesapproximate at best. In a few other cases, anadditional third peak was observed near the pIvalue of zwitterionic compounds, as shown inFigure 2A for sulpiride at pH 9.66. Interestingly,only two peaks were observed at pH values below9.6 and above 10.0, with relative peak areaswithin 1–2% of each other. Analysis by singlecapillary CE with diode array UV detection at pH9.6 found that the middle peak corresponded toDMSO, while the leading and trailing peakspossessed very similar UV spectra. When fittingthe meff versus pH data, the points at pH 9.6 and10.0 noticeably diverged from the best-fit lineapplied to the data (Fig. 2B). Removal of these


Figure 2. (A) Overlay of electropherograms obtained at different pH values for 50 ppmsulpride in 1 mM HCl, 0.1% DMSO. The peak assigned to DMSO is marked with �. Theelectropherograms are offset for clarity. (B) Plot of meff versus pH for sulpiride, assumingthe leading peak at pH 9.66 and the trailing peak at 10.02 correspond to sulpiride.

14 SHALAEVA ET AL.

outlier points led to a better R2 value, increasedreproducibility and improved agreement to lit-erature values. Although still under investiga-tion, we speculate from the above findings that theadditional third peak observed at pH 9.6 may berelated to the presence of multiple stabilizedspecies with slightly different overall charge.

In summary, to maximize the measurementprecision of closely spaced pKa values by CE itis important to utilize evenly spaced buffersof carefully controlled pH and to ensure goodseparation resolution between the compound andneutral marker whenever possible, also by way ofadjusting the vacuum setting.


Identification of Compound Degradation/Impuritiesand Solubility Limits for Aqueous pKa Measurements

A key advantage of CE when measuringcompound pKa values is the ability to separatesample components possessing different mass-to-charge ratios. This ability is of substantial benefitwhen working in a Pharmaceutical Discoverysetting, where the purity and stability of com-pounds has often not been fully assessed. A fewreports have described the application of CE-based pKa measurements to impure and/or labilecompounds. Ornskov et al.30 proposed a strategyfor measuring the pKa values for labile compounds

DOI 10.1002/jps


by CE including the use of a stabilizing samplediluent, electrokinetic sample injection, rapidanalysis times, and characterization of peakcomponents by UV-Vis spectra. In addition tomeasuring the pKa value, Simplıcio et al.32 wereable to examine elimination rates of labilecompounds by CE. The integration of MS detec-tion was also found to be useful when observingcompound impurities.43

When employing multiplexed CE for pKa

measurement, a single fixed UV wavelength isemployed for detection and therefore identifica-tion of sample components by UV-Vis or massspectra is not possible. However, degradation oflabile compounds can often be assessed by dif-ferences in measured meff and migration behavioras shown in Figure 3 for indomethacin, a well-known case used as an example here. Threedistinct analyte peaks were observed over themajority of pH values above pH 3.9 for indo-methacin samples prepared in 1 mM NaOH(Fig. 3). Plots of meff versus pH could be derivedfor the three distinct species and the measuredapparent pKa values (I¼ 50 mM) and predictedcharge valency for the three species (assuming aMW 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 highestMW and therefore the lowest meff indicating thatthe leading analyte peak in Figure 3 is indo-methacin and trailing two analyte peaks aredegradation products. This is indeed the case, asanalysis of indomethacin prepared in water yieldsa single analyte peak corresponding to the leading

Figure 3. Overlay of electropherograms ob10.40, dissolved in different sample matrices(marked by �).

DOI 10.1002/jps

peak with an apparent pKa value and predictedvalency of 4.04 (�1.06). The differences inmeasured pKa values between indomethacinprepared in water and 1 mM NaOH are due topartial peak overlap with the sample degradantsat pH values near the pKa value. Importantly,the ability to predict compound valency permittedthe identification of the intact compound in thepresence of multiple degradation impurities, andall species in this case could be separated andtheir pKa values measured by CE. Such ananalysis is not possible by traditional batchmethods employing potentiometry or spectropho-tometry.

The solubility limitations of many drug-likecompounds also deserve some comments as wetested a number of compounds previously identi-fied as low or sparingly soluble (e.g., amiodarone,tamoxifen, indomethacin, flumequine, furose-mide, ketoconazole) 35,43,62 and found that a largenumber were easily measurable by our methodusing aqueous buffers. However, some compounds(identified by superscript a in Tab. 1) presented achallenge when analyzed at typical workingconcentrations of 50–100 mg/mL. Precipitationof low solubility compounds when employing CEfor pKa measurement is identifiable by significantbroadening of analyte peaks or the appearanceof a plateau in place of the typical Gaussianpeak profile obtained for compounds in solution.Another indication of precipitation is an abruptdisappearance of the analyte peak at a particularpH value. The precipitation of a low solubilitycompound is shown in Figure 4A for the case of

tained for 50 ppm indomethacin at pHcontaining 0.1% DMSO neutral marker


Figure 4. (A) Overlay of electropherograms obtained at pH 9.20 and pH 9.60 foramitriptyline prepared at 50 ppm concentration (top two traces) and diluted 10-fold to5 ppm concentration (bottom two traces), containing 0.1% DMSO neutral marker (�). Theelectropherograms are offset for clarity. (B) Plots of meff versus pH and best-fit lines foramitriptyline analyzed at 50 ppm concentration (&) and diluted 10-fold to 5 ppmconcentration (~). Precipitation occurring at pH 9.20 and above in the more concen-trated sample resulted in an underestimation of the pKa value.

16 SHALAEVA ET AL.

amitriptyline, which possesses a reported aqueousintrinsic solubility of 1.8 mg/mL (6.5 mM).5 Theresult is a general underestimation of pKa anddilution or use of cosolvent are required to solvethe problem, as shown still for amitriptyline inFigure 4B, but its assessment a priori is difficulteven with logD data.

Potential Challenges with AqueouspKa Measurements

In addition to solubility issues encountered duringaqueous pKa analysis, a few compounds exhibited


a particular behavior during measurement, forexample chloroquine and tetracycline in certainpH ranges, and yielding broad peaks.

The anomalous results for chloroquine andtetracycline may be due either to an interactionwith silanol groups on the capillary wall or withthe phosphate buffer ions. It has been reportedthat tetracycline has a strong affinity for silanolgroups present in HPLC stationary phases.63

Additionally, interactions between certain multi-ply charged and/or hydrophobic basic compoundswith the negatively charged capillary wall hasbeen previously discussed.64 In these cases alter-

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native buffers have been previously utilized forCE-based pKa measurements, including MES,HEPES, MOPS, TAPS, AMPSO, CAPS, etc.21,22 Inaddition, certain amine containing buffers such asMES and HEPES are known to interact with thecapillary walls, effectively competing with anddiminishing analyte adsorption.64

Methanol/Water Cosolvent pKa Measurements

Comparison of Cosolvent pKa Measurementsto Literature Values

Several compounds in the test set (e.g., amiodar-one and chlorpromazine) were found to precipitateduring aqueous measurements, even when dilut-ed to the detection limits of the more sensitivecePRO instrument (5–10 mg/mL, correspondingto 17–34 mM for MW¼ 300). To measure thepKa values for compounds with low aqueous

Table 3. Summary of pKa Values Measured by 96-CapillarYasuda–Shedlovsky Extrapolation to 0% Cosolvent and a C



Literature Difference

1 Amiodaronea 8.71 8.88 �0.172 Amitriptyline 9.49 9.41 0.083 Bifonazole 6.11 5.80 0.314 Chlorpromazine 9.16 9.25 �0.095 Clomipramine 9.36 9.28 0.086 Clotrimazole 5.83 5.89 �0.067 Desipramine 10.30 10.27 0.038 Flufenamic Acid 4.01 4.02 �0.019 Imipramine 9.50 9.50 0.00

10 Mebendazole 3.20 3.43 �0.239.64 9.93 �0.29

11 Miconazole 6.38 6.35 0.0312 Nefazodone 6.65 n/a13 Nicardipine 7.12 7.34 �0.2214 Nortriptyline 10.02 10.11 �0.0915 Promazine 9.38 9.11 0.2716 Promethazine 8.71 8.93 �0.2217 Quinacrine 7.29 7.74 �0.45

9.87 10.08 �0.2118 Quinine 4.39 4.20 0.19

8.53 8.51 0.0219 Tamoxifenb 8.60 8.60 0.0020 Terfenadineb 9.40 9.54 �0.1421 Trazodone 6.60 6.75 �0.1522 Trimipramine 9.25 9.25 0.0023 Verapamil 8.50 8.81 �0.31

aDue to precipitation, only values from 50% to 60% (v/v) methanbDue to precipitation, only values from 40% to 60% (v/v) methancPotentiometric measurement performed by pION, Inc.dPotentiometric measurement Pfizer, Sandwich Laboratories.

DOI 10.1002/jps

solubility a method employing methanol/watercosolvent buffers was developed. The apparentswpKa values (I¼ 50 mM) were measured over fourdecreasing percentages of methanol cosolvent(60%, 50%, 40%, 30%, v/v), converted to apparentsspKa values using the appropriate d term viaEq. (3), and extrapolated to 0% cosolvent to yieldthe apparent w

wpKa value using the widelyaccepted Y–S method.9,56,57,59 In addition to theaforementioned low solubility compounds, thedata set used to evaluate the cosolvent methodincluded 16 additional compounds previouslymeasured with aqueous buffers to further assessthe approach. The data set consisted primarilyof monobasic compounds with two dibasic com-pounds (quinacrine and quinine), one acidiccompound (flufenamic acid) and one monoacidic/monobasic zwitterion (mebendazole).

Table 3 lists the average extrapolated appa-rent w

wpKa values at I¼ 50 mM measured by

y Array CE Using Mixed Methanol/Water Buffers withomparison to Literature Values

pKa RangeLiterature References

8.7–9.06 5,739.32–9.49 5,45,51,71,725.72–5.88 77 c

9.15–9.38 43,67,68,69,109,1219.17–9.38 71 c

5.48–6.3 85 c

10.16–10.65 5,35,51,72,74,863.63–4.27 5,27,33,679.34–9.66 5,34,35,43,49,51,72 c

3.43 439.93

6.07–6.63 5,28n/a

7.17–7.41 28,109,12210.02–10.19 5,45,49,518.92–9.40 72,109,1218.62–9.10 5,68,72,1097.73–7.74 68,1239.97–10.183.95–4.46 13,14,21,26,28,29,33,35,43,45,59,68,82,918.35–8.608.48–8.71 5,439.21–9.86 5,436.69–6.79 33,49 c

9.15–9.37 49 cd

8.66–9.07 5,22,27,28,35,58,74,109

ol were used.ol were used.


18 SHALAEVA ET AL.

multiplexed CE using the cosolvent method withthe Y–S extrapolation along with a comparisonto published literature pKa data. Literature datawere treated in a similar manner as theaqueous pKa measurement comparisons. Themajority of literature data for the relatively lowsolubility compounds in the data set wereobtained using Y–S cosolvent extrapolation meth-ods. The average difference between the cosolventextrapolated pKa values in this work and theaverage literature pKa values was found to be�0.07 U (average absolute error of 0.15 U). Acorrelation plot of the data from Table 3 yieldeda slope of 1.005 with an intercept of þ0.027(R2¼ 0.992) (data not shown). Taken together,these results indicate a good level of agreementbetween the cosolvent method of this workcompared to previous studies.

We note that it was not possible to analyze a fewcompounds over the entire cosolvent range from30% (v/v) to 60% (v/v) methanol due to precipita-tion during the analysis. Tamoxifen and terfena-dine were found to precipitate at 30% (v/v)methanol, while amiodarone precipitated up to40% (v/v) methanol. With a reported aqueousintrinsic solubility of only 0.005 mg/mL (8 nM),5

amiodarone represents an extreme lower limitof aqueous solubility and yet its pKa could bedetermined over at least two percentages ofmethanol cosolvent.

Figure 5 shows some representative Y–S extra-polation plots of 1/e (�1000) versus (apparentsspKa þ log[H2O]) for several different compounds.The plots display good linearity (R2� 0.97, exceptfor the upper pKa of mebendazole where R2¼ 0.75)

Figure 5. Representative Yasuda–Shedlovsvarious compounds.


and varying slopes depending upon the compoundstructure and ionization type, with an observedtrend of acidic compounds showing a positive slopeand basic compounds showing a negative slope,consistent as well with earlier reports whichfound that basic ionizable groups produce nega-tive slopes while acidic groups possess positiveslopes when performing Y–S extrapolations frommethanol cosolvent mixtures.9,59 The decreasein pKa value for bases and increase in pKa valuefor acids with increasing methanol content resultsfrom suppression of ionization upon the corre-sponding decrease in the dielectric constant of themedium. The slopes for the Y–S plots in this workvaried somewhat between compounds dependingupon the type of ionizable group. A detailedanalysis of Y–S plot slope variations is beyond thescope of this study, but has been previouslyattributed to differences in both the chemicaland solvation structure of compounds.59

Interlaboratory Comparisons ofCosolvent pKa Measurements

Table 4 shows a comparison between laboratoriesof the extrapolated apparent w

wpKa values (I¼50 mM) obtained using the multiplexed CEcosolvent method. The absolute average differ-ence between measured pKa values was 0.12 U.The average repeatability (SD) was comparablebetween instruments (cePRO� 0.06, MCE 2000�0.09). Again, in both cases measurements wereacquired over several months with at least twodifferently prepared lots of cosolvent buffers tobetter simulate long-term repeatability.

ky pKa extrapolation plots obtained for

DOI 10.1002/jps

Table 4. Comparison of pKa Values Measured by Two Laboratories/InstrumentsUsing Mixed Cosolvent Buffers

Compound pKa by cePRO SDpKa by

MCE2000 SD Difference

Amiodaronea 8.78 0.08 8.64 0.15 0.14Amitriptyline 9.49 0.16Bifonazole 6.18 0.02 6.01 0.03 0.17Chlorpromazine 9.21 0.04 9.10 0.07 0.11Clomipramine 9.35 0.05 9.37 0.12 �0.02Clotrimazole 5.87 0.03 5.78 0.09 0.09Desipramine 10.30 0.20Flufenamic acid 4.01 0.04Imipramine 9.50 0.09 9.51 0.03 �0.01Mebendazole 3.20 0.06

9.64 0.14Miconazole 6.40 0.06 6.30 0.11 0.10Nefazodone 6.67 0.05 6.63 0.01 0.04Nicardipine 7.26 0.02 6.98 0.07 0.28Nortriptyline 10.03 0.06 10.00 0.04 0.03Promazine 9.47 0.03 9.32 0.08 0.15Promethazine 8.71 0.13 8.71 0.03 0.00Quinacrine 7.29 0.06

9.98 0.12 9.75 0.16 0.23Quinine 4.41 0.12 4.36 0.13 0.05

8.64 0.05 8.42 0.05 0.22Tamoxifenb 8.62 0.10 8.57 0.19 0.05Terfenadineb 9.52 0.05 9.27 0.08 0.25Trazodone 6.67 0.03 6.52 0.08 0.15Trimipramine 9.29 0.08 9.16 0.05 0.13Verapamil 8.68 0.04 8.35 0.09 0.33

aDue to precipitation, only values from 50% to 60% (v/v) methanol were used.bDue to precipitation, only values from 40% to 60% (v/v) methanol were used.


From Table 4 it can be observed that the Y–Sextrapolated results from the MCE 2000 are ingeneral slightly lower than the results from themore sensitive cePRO instrument. One possibleexplanation for this result is the longer time to fillthe inlet pH buffers into the capillary array andperform the analysis on the MCE 2000 instru-ment. The buffer plates used in both instrumentsare not sealed to avoid damage to the capillary tipswhich could result from the repetitive piercing ofwell caps. In addition, in the case of the MCE 2000instrument a few results were obtained fromthe repeated use of the same prepared cosolventbuffer plate. As a result, some evaporation likelyoccurred from the buffer plate between analyses,particularly at higher % methanol contents. Anydecrease in methanol concentration would leadto an increase in measured apparent s

wpKa valuesfor basic compounds, with greater evaporationoccurring at higher methanol content. Indeed,a comparison of the results for nicardipine

DOI 10.1002/jps

and verapamil between labs revealed that theaverage apparent s

wpKa values at 60% (v/v)methanol were higher by 0.11 and 0.07 U,respectively, on the MCE 2000 instrument com-pared to the cePRO instrument. For this reasonthe repetitive use of cosolvent buffer plates wasdiscontinued.

It was also observed in the course of this workthat a measurable increase in temperature occursduring the titration to the target s

wpH value whenpreparing the cosolvent buffers. Upon cooling toroom temperature, pH shifts of up to 0.1 U wereobserved for a few of the cosolvent buffers. Thepreparation procedure has since been modified toallow cooling of the solution to room temperatureprior to final buffer pH adjustment. This also mayaccount for some of the observed differences inresults.

The more automated cePRO instrument shouldat any rate eliminate most of the instrument-related experimental variations due to manual


20 SHALAEVA ET AL.

sample/buffer plate exchange or different capil-lary filling and analysis times.

Comparisons of Aqueous and Cosolvent pKa Results

Table 5 shows a comparison between the averageextrapolated apparent w

wpKa values measuredwith the multiplexed CE cosolvent method andthe average apparent pKa values obtained withaqueous buffers. The average deviation of the Y–Sextrapolations from aqueous measurements forbasic pKa values was �0.17 U (n¼ 14) and foracidic pKa values was þ0.06 (n¼ 2) with an over-all absolute average error of 0.17. Although arelatively small number of compounds werecompared, this result is again in agreement withearlier studies which found that the Y–S extra-polation method in general yielded linear plotswith a slight underestimation of pKa values forbasic compounds and a slight overestimationof pKa values for acidic compounds.59

Further Observations on CosolventpKa Measurements

Overall, the multiplexed CE cosolvent methoddescribed above was able to successfully measurethe pKa values for low solubility compounds withgood agreement to previous literature data. Thetotal time to measure the extrapolated w

wpKa

values for four compounds over 24 pH points

Table 5. A Comparison Between the AveraObtained with the Cosolvent Method via Y–SApparent pKa Values Obtained with Aqueous

CompoundpKa, Average,

Y–S Extrapolation

Amitriptyline 9.49Bifonazole 6.10Clomipramine 9.36Clotrimazole 5.83Desipramine 10.30Flufenamic acid 4.02Imipramine 9.51Mebendazole 3.20

9.64Nortriptyline 10.02Promethazine 8.71Quinine 4.39

8.53Trazodone 6.52Trimipramine 9.23Verapamil 8.52


and four % cosolvent compositions was approxi-mately 2 h. This is a significant improvement insample throughput over the traditional cosolventpotentiometric method, which requires 2–3 h formeasurement of a single compound over 3–4%cosolvent compositions, and single capillary CEmethods which would require many hours tomeasure a single pKa value over several differentcosolvent compositions.

We found that it was important to preparethe cosolvent buffer plates immediately prior toperforming the cosolvent experiments, in order tominimize any effects of buffer evaporation on theexperiment. Using a repeater pipette, it is possibleto prepare the 24 point buffer plates in approxi-mately 10 min. This preparation time can beeliminated through the use of premade, sealedbuffer plates which are currently under develop-ment. For the same reason the cosolvent bufferplates should not be reused for multiple experi-ments.

We also observed that it is beneficial to preparesamples for the cosolvent measurements in 60%(v/v) methanol to better match the composition ofthe buffer solutions. This is due to an improve-ment of the DMSO (the neutral marker employedin this work) peak shape, non-Gaussian particu-larly at higher pH values and fully aqueousdiluents, which could affect the proper assign-ment of the migration time. The use of a samplediluent which contains a similar or higher

ge Extrapolated Apparent wwpKa Values

Extrapolation and the AverageBuffers

pKa, Average,Aqueous Difference

9.51 �0.026.29 �0.199.57 �0.215.99 �0.16

10.42 �0.123.97 0.059.58 �0.073.27 �0.079.56 0.08

10.29 �0.279.05 �0.344.29 0.108.53 0.006.80 �0.289.56 �0.338.93 �0.41

DOI 10.1002/jps


proportion of methanol compared to the run buffergreatly improved the DMSO peak shape.

When analyzing the cosolvent titration curvedata for several basic compounds with relativelyhigh pKa values (e.g., clomipramine, nortripty-line, imipramine) there was a noticeable ‘‘jump’’in the effective mobility upon increasing theswpH value and switching between phosphate(swpH¼ 8.40) and borate (s

wpH¼ 8.80) cosolventbuffers. This result is consistent with a recentreport by de Nogales and coworkers who observedthat the measured effective mobility was lower forseveral hydrophobic basic drugs when 50% (v/v)methanol/water cosolvent buffers were preparedfrom phosphate and/or borate as compared toother common buffers such as tris, ammonia,butylamine, or ethanolamine.65 In the case ofphosphate, this phenomenon was ascribed tospecific interactions taking place between thenegatively charged phosphate buffer ions andthe positively charged analyte. Comparison of themeasured apparent s

spKa values (I¼ 50 mM) foramiodarone, trimipramine, imipramine, and nor-triptyline at 50% (v/v) methanol–water to valuesmeasured by de Nogales and coworkers found thaton average the values measured in this work areapproximately 0.1 pH units lower, indicating thatthe use of phosphate and/or borate buffers mayhave resulted in a slight underestimation of themeasured pKa values. Further work is needed tomore fully elucidate the effects of buffer type onthe measured mobility of different compounds incosolvent mixtures. A potentially useful applica-tion of this observation is that it may provide atool to directly identify potential buffer-analyteinteractions and their effect on the measuredpKa value.

CONCLUSION

We have reported a good and fairly rapid methodfor the measurement of pKa values by multiplexedCE which has been shown to yield accurate resultswith low inter- and intra-laboratory variability.The method is capable of handling measurementsmade using water soluble compounds and lowsolubility compounds alike, via the use of a cosol-vent approach we have optimized and adoptedafter extensive testing.

Our stated goals included the study of theaspects mentioned above and, among others, theuse of an enhanced set of buffers to be able todiscern closely spaced pKa values, often missed by

DOI 10.1002/jps

other types of measurements and, in some cases,reported as such in the literature. We havediscussed prior efforts and used quality literaturedata, from thorough searches and data analysis,to benchmark our results. We have kept in mindall along the application of the method in anindustrial discovery setting and offered practicalconsiderations and clues, based on extensiveexperience with the method by two laboratories,on issues one may encounter with stability andlow solubility of compounds and how to recognizeand overcome some of them. We have also shownthat the use of mass spectrometric detection is notwarranted in most cases as we were able tosuccessfully measure extremely low solubilitycompounds.

We believe this method is readily applicable andcapable of yielding accurate results and also offersadded advantages in terms of the low amount ofcompound needed. Importantly, the multiplexedCE method is capable of recognizing the numberof pKa values present in a compound by relatingcompound MW, effective mobility, and charge.Finally we also note, among its potential advan-tages over other methods, the possibility toidentify potential solubility and/or stability and/or buffer-solute interaction issues in addition tomeasuring the pKa value.

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