Trends in Analytical Chemistry, Vol. 26, No.

11, 2007

Trends

Determination of pKa values of active pharmaceutical ingredients
´ , Alka J.M. Horvat, Dragana Mutavdz ´ Pavlovic ´, Sandra Babic ˇ ic ˇtelan-Macan Marija Kas
The acid dissociation constant is an important physicochemical parameter of a substance, and knowledge of it is of fundamental importance in a wide range of applications and research areas. We present a critical review of published methods and approaches for the determination of acid dissociation constants, with a special emphasis on the pKa values of active pharmaceutical ingredients. ª 2007 Elsevier Ltd. All rights reserved.
Keywords: Active pharmaceutical ingredient; Capillary electrophoresis; Acid dissociation constant; Liquid chromatography; Potentiometry; Spectrophotometry Abbreviations: l, Electrophoretic mobility; API, Active pharmaceutical ingredient; CE, Capillary electrophoresis; LC, Liquid chromatography; k, Retention factor; Ka, Acid dissociation constant; pKa, Negative decadic logarithm of acid dissociation constant; NMR, Nuclear magnetic resonance; UV-VIS, Ultraviolet-visible spectrometry

1. Introduction
´ *, Sandra Babic Alka J.M. Horvat, ´ Pavlovic ´, ˇ ic Dragana Mutavdz ˇtelan-Macan Marija Kas Laboratory for Analytical Chemistry, Faculty of Chemical Engineering and Technology ´ ev University of Zagreb, Marulic trg 19, Zagreb, Croatia

*

Corresponding author. Tel.: +385 1 4597 205; Fax: +385 1 4597 250; E-mail: sandra.babic@fkit.hr

Active pharmaceutical ingredients (APIs) are among the so-called ‘‘emerging’’ contaminants that have caused great concern in recent years. They are continually being introduced in the environment mainly as a result of manufacturing processes, or disposal of unused or expired products and excreta. Many of these substances or their bioactive metabolites end up in waters, soils and sediments, where they can accumulate and induce adverse effects in terrestrial or aquatic organisms [1,2]. Relevant processes regarding pharmaceuticals in the environment include sorption to soils and sediments, complexation with metals and organics, chemical oxidation with natural or water-treatment oxidants, photolysis, volatilization, biodegradation, purification, and analytical isolation. Information on the physical and chemical properties (e.g., the octanol/

water partition coefficient, dissociation constants, vapor pressure or HenryÕs Law constant of an active pharmaceutical ingredient) may help to determine whether a compound is likely to concentrate in the aquatic, terrestrial, or atmospheric environment. Since most APIs have acidic and/or basic functionalities, their ionization state is controlled by both solution pH and acidic dissociation constants (i.e. Ka values). These different chemical species (cationic, neutral, or anionic) often have vastly different properties with respect to water solubility, volatility, UV absorption, and reactivity with chemical oxidants. The ionized form is usually more water soluble, while the neutral form is more lipophilic and has higher membrane permeability. The extent of ionization is one of several cardinal properties used to estimate the absorption, distribution, metabolism and excretion of compounds in biological systems and the environment. From dissociation constants, the major species of pharmaceuticals present in the environment (usually in neutral pH range) can be estimated [3,4]. Consequently, it is very important to know dissociation constants for environmentally relevant APIs in order to estimate their occurrence, fate and effects. Knowledge of pKa values as a function of solvent composition is also useful in applying liquid chromatography (LC) or capillary electrophoresis (CE) for the separation of ionizable compounds. The chromatographic retention and electrophoretic behavior of ionizable compounds 1043

0165-9936/$ - see front matter ª 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.trac.2007.09.004

as in potentiometry. potentiometric titration was the standard method for pKa measurement.13]. automated instruments. and leastsquares non-linear regression [3. The absorption spectra of the sample changes during the course of the titration to reflect the concentration of neutral and ionized species present. Using a proper experimental arrangement. In the past decade. Potentiometric titration is a high-precision technique for determining the pKa values of substances. pKa determination 2. 2. some alternative techniques for dissociation-constant determination. potentiometry [3. In a potentiometric titration. but they require different spectra for different species and reagents must be pure. Spectrophotometric methods An alternative to potentiometric titration is UV–VIS spectrophotometry because it can handle compounds with lower solubility and lower sample concentrations.11] and UV–VIS absorption spectrometry [5] have been the most useful techniques for the determination of equilibrium constants. 26.8] and CE [9]. Moreover. the pKa has become of great importance because the transport of drugs into cells and across other membranes is a function of physicochemical properties. a compound must contain a UV-active chromophore close enough to the site of the acid–base function in the molecule. its shortcomings include the requirements to use a milligram of pure compounds and a mixture of aqueous buffers [10]. absorption.2. Particularly for developing new APIs. based on separation methods (e. Target-factor analysis has been applied to deduce pKa values from the multi-wavelength UV absorption data recorded at different pH values [9]. Moreover. The largest change in absorbance occurs at the pH corresponding to a pKa value. It is commonly used due its accuracy and the commercial availability of fast. If an equilibrium is sufficiently fast in comparison with the separation process. To use this method. without a sample present.5]. Traditionally. a sample is titrated with acid or base using a pH electrode to monitor the course of titration. Spectral data are recorded continuously during the course of titration by a diode-array spectrometer. Potentiometric titration In the past. acid–base. Analysis methods commonly used to derive pKas from titration curves include GranÕs plot [10]. after which the pKa is determined. However. However. second-derivative (D2pH/DV2) [3]. the absorption spectra of individual species must be characterized beforehand and the molar absorptivities of protonated and deprotonated species are thus required. 2007 strongly depend on the pKa of the compound and the mobile-phase pH [5. Spectrophotometric methods offer excellent precision. respectively.com/locate/trac . 2. then retention characteristics in chromatography (retention factors) or migration characteristics in electrophoresis (effective mobilities) may be expressed as functions of the composition of the mobile phase or background electrolyte. Chemical interactions (e. since the spectra of impurities can overlap with those corresponding to the solutes of interest [5. so a multi-wavelength spectrophotometric approach has been developed to determine acid dissociation.g.7. due to their accuracy and reproducibility. These changes are usually identified from the first derivative of the absorbance against time plot or from overlay plots of the different spectra. No. However. and of the pKa of the drugs [10].10.5]. these techniques can be used advantageously to study chemical equilibria affecting separations.. The pKa value is calculated from the change in shape of the titration curve compared with that of blank titration 1044 http://www. The determination of pKa values by UV–VIS assumes that the solute of interest is pure or that its impurities do not absorb in the UV–VIS range. The main advantage is higher sensitivity (>10À6 M) to compounds with favourable molar absorption coefficients. In most of these methods. 11. distribution.6]. the dependencies of retention (migration) characteristics on the mobile phase (background electrolyte) composition can be measured and utilized to calculate equilibrium constants for equilibriums taking place in the mobile phase (background electrolyte) [12]. carbonate-free solutions must be prepared laboriously [3.g. pKa values can also be predicted by computational methods on the basis of molecular structure.elsevier. a physical property of an analyte is measured as a function of the pH of a solution and resulting data are used for the determination of dissociation constants. Traditionally. in such a case. LC [12] and CE [4]) have been developed. Solutions of at least 10À4 M are required in order to detect a significant change in shape of the titration curve.1. Vol. spectral data at a single analytical wavelength are acquired from a sample in a series of buffer solutions with known pH values.Trends Trends in Analytical Chemistry. Satisfactory knowledge of the acid–base behavior of substances in hydro-organic media is therefore essential to optimize analytical procedures for the separation of ionizable compounds by LC [7. complex-forming. ion association and other equilibria) are widely exploited to improve separation efficiency and selectivity in LC as well as in electrophoresis. the acid–base property of a drug molecule is the key parameter for drug development because it governs solubility. especially for measurements at neutral-to-high pH. There are several methods for the determination of dissociation constants.8. To avoid errors. metabolism and elimination. These measurements are non-trivial if acid-base equilibria comprise more than two ionization steps or if reacting components are not stable within two pH units of the pKa value..

NMR titration NMR-pH titration is also an excellent technique for determining pKa values as the protonation of a basic site leads to electronic deshielding effects on the adjacent NMR-active nuclei. Intermediate mobility is therefore a function of dissociation equilibrium. Capillary electrophoresis (CE) The determination of pKa values by CE is based on observation of the effective mobility of an ionizable compound in a series of electrolyte solutions of constant ionic strength and various pHs. (1) can be written as: Ka kHA þ kAÀ ½H þŠ k¼ or Ka 1 þ ½H þ Š kHA Á 10ÀpH þ kAÀ Á 10ÀpK a ð4 Þ k¼ 10ÀpH þ 10ÀpK a This equation is related to the chromatographic retention of a monoprotic substance and the pH of the mobile phase.i ð5 Þ i ð1 Þ The molar fractions can be expressed as a function of the dissociation constants and molar concentration of the hydrogen ion or as a function of pH and pKa values: xHA ¼ xAÀ ½H þ Š ½H Š þ K a Ka ¼ þ ½H Š þ K a þ or xHA ¼ or xAÀ 10ðpKa ÀpHÞ 1 þ 10ðpKa ÀpHÞ 1 ¼ 1 þ 10ðpKa ÀpHÞ ð2 Þ ð3 Þ Substituting the terms xi.14]. Vol. and can be used to determine pKa values and also to predict the chromatographic behavior of substances. This method does not include measuring solute or titrant concentrations. Liquid chromatography (LC) The determination of pKa values by means of LC is based on the relationships between capacity factors and the pH of the mobile phase. The fraction of acid present as the anionic form is given by Eq. calculation is straightforward and independent of solute purity. for the general case. 26.3. To our knowledge and according to available references. No. However. therefore. a solute has zero mobility while its fully ionized state has maximum mobility. where tM is the hold-up time. where xi is the fraction of the monovalent acid present as the anionic form and lep. Activity coefficients for ions in a dilute electrolyte solution at 25°C can be estimated according to classical Debye–Hu ¨ ckel theory. are expected to reflect the fractional protonation of each basic group of a molecule. For monoprotic acid. corrections for activities are not shown. so. Eq.i.i is the electrophoretic mobility of an anion. this method is perfectly adequate for this purpose. studied samples do not need to be pure and poor water solubility is not a serious drawback. 2007 Trends 2. (4) [6.Trends in Analytical Chemistry. just retention times. the observed capacity factors at different pH values can be described as a function of the capacity factors of neutral and anionic species (kHA and kAÀ) and their corresponding molar fractions (xHA and xAÀ) [6]: X k ¼ xHA kHA þ xAÀ kAÀ or more generally : k ¼ xi k: i LC is used as a powerful technique for the determination of dissociation constants. retention time values.com/locate/trac 1045 . due to the large retention times observed. pKa values are determined by performing a non-linear fit to Eq. which is determined for each mobilephase composition and pH studied. the standard deviations of pKa values are higher than those obtained by potentiometric or spectrophotometric methods [5]. where xi is the mole fraction of species i with electrophoretic mobility lep. One of the most important disadvantages of the LC method is that the pH of the mobile phase and. In its uncharged state. 11. 2.12. For simplicity. Effective mobility is calculated from the experimentally determined migration time of an analyte and the electroosmotic flow at different pH values using the following equation: leff LLeff ¼ V  1 1 À tm teo  ð7 Þ http://www. as the main objective of pKa determination by LC is the optimization of chromatographic separations.elsevier. as a function of pH. effective mobility is given by: leff ¼ lep Á 10ÀpK a 10ÀpK a þ 10ÀpH ð6 Þ pKa values are obtained by fitting effective mobility as a function of pH to a suitable model for the number of ionizable groups. Nevertheless. as it requires only a small quantity of compounds. the range of pKa values that can be determined are limited by the stability of the column package. Retention factors are calculated from k = (tR À tM)/tM.i. NMR titrations have not yet been applied to the pKa determination of APIs. (3). are determined. for a monovalent weak acid. Using this relation. Moreover. tR. and. the effective mobility is given by leff = xi Æ lep. The on-line coupling of a potentiometric titrator with an NMR spectrometer results in a powerful hyphenated technique called NMRcontrolled titration [10.4.5. the effective mobility is given by: X leff ¼ xi lep. it is not easy to determine pKa values in water and aqueous–organic mixtures with low contents of organic solvent. so the average chemical shifts of all the measurable NMR-active nuclei. There is no restriction on the number of ionization equilibria involved.15]. Also. 2. For each compound and for every mobile phase composition and pH considered.

08 ± 0.30.01.27 1.13 ± 0.06 ± 0.24 Calculated (SPARC) [23] Sulfamerazine O S H2N Sulfamethazine O N N H 2.06 1.95 ± 0.87. 5. 7.30 ± 0.55.49 ± 0. 7. [19] [37] [37] O S O N H O <2.42 2.elsevier.90 ± 0. Dissociation constants of sulfonamides Compound Sulfacetamide Chemical structure pKa Method CE-MS (positive detection mode) Multi wavelength spectrophotometry UV/pH Ref.28 CE CE [36] [25] N OCH3 O S H2N O N H N N OCH3 2. 5. Vol.09 1.65 2.05 CH3 H2N Sulfachloropyridazine O S H2N O N N H N Cl 1.04.45 ± 0.01. 6.01 7.30. 6.05 UV/pH titration CE Potentiometry [13] [25] [3] N CH3 O S H2N O N N H 2.17. No.30.80 ± 0.77 2.Trends Trends in Analytical Chemistry. 7.76 ± 0. 5.13 UV/pH titration CE CE Potentiometry [13] [36] [25] [3] N CH3 1046 http://www. 6.01 1.37 ± 0.86 Potentiometry CE [3] [25] Sulfaguanidine O S H2N O N H NH NH2 1.01 2.10. 26.com/locate/trac . 5.28.40 Potentiometry CE [3] [25] Sulfadiazine O S H2N Sulfadimethoxine O N N H 6. 5. 6.90.43 2. 6.30. 11.22 ± 0. 11.87 ± 0. 2007 Table 1.5.22 ± 0.07 ± 0.49 ± 0. 5.11.

Electrolyte solutions of a different pH with a low and constant ionic strength as well as effective column thermostatting are therefore required for pKa measurements.01 ± 0. [3] O S H2N Sulfamethoxazole O N N H N S H CH3 1. pH.10.57 1. The most popular buffers for CE are phosphate.20]. since CE is a separation technique that can handle impure samples. and teo is the migration time of a neutral marker compound that is carried through the column by the electroosmotic flow. No. acceptable shelf life. it is only necessary to determine pH-dependent mobilities. and availability in a high-purity form. only a few nanograms or less are commonly used for analysis. A recent review summarized numerous pKa determinations by CE [4].Trends in Analytical Chemistry.. with this technique.g.. pKa values may also be determined for relatively unstable compounds [17. 5.  the procedure does not require measurement of solute concentration. 26. V is the applied voltage. temperature and viscosity) [16]. acetone or dimethyl sulfoxide [8.60 ± 0. Suitable marker compounds for estimating teo are methanol. The models for weak acids and bases containing up to three ionization centers have been summarized [4]. reasonable water solubility.08. 11.07 2.09 CE CE Potentiometry [36] [25] [3] where L is the total capillary length. 2. 5.12. ionic strength. borate and zwitterionic compounds (GoodÕs buffers) [16].16].com/locate/trac 1047 .30.04 CH3 O S H2N O N H OH N 5. Vol. The CE technique is expected to be especially useful if the amount of compound is very limited because.elsevier.:  concentration is limited by only the limit of detection of a compound. 7. acetate.18]. The effective electrophoretic mobility of weak acids and bases is strongly influenced by environmental factors that affect underlying equilibrium constants (e.24 2. Leff is the effective capillary length (to the detector).04 CE CE Potentiometry [36] [25] [3] Sulfathiazole O S H2N O N H N S 7.11 ± 0. and temperature) as well as variables that affect ion mobility (e.29 ± 0.g. a new procedure has been developed when LC and CE methodologies are used for pKa determination in combination with a diode-array detector http://www. For CE. ionic strength. Desirable buffer properties for the measurement of pKa values by CE include detector compatibility (usually low UV absorbing). tm is the migration time of a solute. The high-throughput screening method (simultaneously determining more than 50 compounds in a single sample) has been successfully developed within an advanced CE technique using mass spectroscopy detection [19.6. mesityl oxide.g. and.65 1.83. Instruments are highly automated and require little or no modification for highthroughput applications. 7.85 ± 0.30.30. 5.86 ± 0. Combined methods In recent years. 2007 Trends Table 1 (continued) Compound Sulfamethizole Chemical structure pKa Method Potentiometry Ref. and that has several advantages. e.

7.97 ± 0. 9.00 3.79. 5.55 Potentiometry Calculated (SPARC) [3] [22] NH2 OH OH O OH O O 1048 http://www. 2007 Table 2. 8.59.30 Potentiometry [3] OH OH O OH O O Meclocycline Cl CH2 H3C OH CH3 N OH 4. 7.23 ± 0.46 CE-MS (positive detection mode) Potentiometry Multiwavelength spectrophotometry Calculated (SPARC) [19] [3] [37] [22] NH2 OH OH O OH O O Tetracycline HO CH3 H H3C N H CH3 OH 3. 7.30 3.05.46 ± 0.30. 8.94 Method Potentiometry Calculcated (SPARC) Ref. 9. 7. 7.03.32 ± 0. 5.93 Potentiometry Calculated (SPARC) [3] [22] NH2 OH OH O OH O O Doxycycline CH3 H3C OH N CH3 OH N NH 3.01.com/locate/trac .30 3.02 3. 9.02 ± 0.36 ± 0. 7. 9.37 ± 0.82 ± 0.15. 5.33 ± 0. [3] [22] NH2 OH OH O OH O O Demeclocycline Cl OH H H3C N H CH3 OH 3.41.78 ± 0. 7.99.30.15 ± 0.39.30.22 ± 0.87 ± 0.55 ± 0. 26.02. 8. No.22 ± 0.79. 6.58 ± 0.30.04.30 3.01. 8.elsevier.03.30.95.59 ± 0.53.Trends Trends in Analytical Chemistry. Vol.33 ± 0. 5. 7.02. 8.30.94 ± 0. 11.30 Potentiometry [3] NH2 OH OH O OH O O Oxytetracycline HO CH3 H H3C OH H CH3 N OH 3. 9.44 ± 0.05 ± 0. Dissociation constants of tetracyclines Compound Chlortetracycline Cl HO CH3 H Chemical structure H3C N H OH CH3 pKa 3.30 3.

19 ± 0.25.05 ± 0.02.68. 6. 2007 Trends Table 3.43.01.09.42 ± 0.30 5.03 7.05. 9.18.24 ± 0. 8.26 ± 0.86 ± 0.14 ± 0.50 5. 8.24 ± 0.02 6.90 ± 0. 8.05 Danofloxacin O F N N N COOH Difloxacin O F N N N COOH 5. 6. 3.88 ± 0.56 ± 0.elsevier.04. 8.07 6.28 ± 0. 7.68 ± 0. 8.59 ± 0.91 ±0.15.80 ± 0.68.30.86 ± 0. No.07.30.73 ± 0.12 ± 0. 8. 6. 10.33 ± 0.04 5.com/locate/trac 1049 .01.05. 8.07 ± 0.74 CE CE-DAD Calculated (SPARC) [8] [8] [22] F Enoxacin O F COOH 6. 8. 8.35 ± 0. 6.05 6.85 ± 0.58 ± 0.80 ± 0.75 Potentiomtry CE Calculated (SPARC) [31] [30] [22] N N N Enrofloxacin O F N N CH3CH2 N 5.01.42 CE CE-DAD Potentiometry CE UV/pH Calculated (SPARC) [8] [8] [3] [41] [42] [22] (continued on next page) http://www. 6. 7.62 5.87 7. 11.24 ± 0.66 ± 0.63 6. 8.32 ± 0. 8.70 ± 0.11 6.04 6.06 5.01 ±0. [13] [13] [31] [8] [8] [3] [30] [39] [22] [8] [8] O F COOH N HN N 6. 26.74 ± 0.11 6.07 6. 8. Vol.89 ± 0.86 ± 0.95 ± 0.02.84 ± 0. 6.13.05. 8.56 ± 0.30 8. 8.05 6.49.03. Dissociation constants of quinolones Compound Ciprofloxacin Chemical structure pKa Method UV/pH titration (MDM-water) UV/pH titration (MeOH-water) Potentiometry CE CE-DAD Potentiometry CE Potentiometry/fluorimetry Calculated (SPARC) CE CE-DAD Ref. 7.00.09.01.08.25 ± 0.04 3.05 ± 0.06. 7.09 ± 0.29 ± 0.05.07 7. 6.15.Trends in Analytical Chemistry. 5.

00 6.00 ± 0.02 ± 0. 6.12. Vol.22 ± 0.00 5.45 ± 0.25 ± 0. 8.10.38 5.10. 8.46. [31] [40] [22] O F N H3C N F F N COOH 5.00 ± 0.13 CE CE-DAD [8] [8] Nalidixic acid O COOH 5.elsevier. 2007 Table 3 (continued) Compound Fleroxacin Chemical structure pKa Method Potentiometry Potentiometry/fluorimetry Calculated (SPARC) Ref.22.10.19.Trends Trends in Analytical Chemistry. 26. 8.62 ± 0.00 ± 0. 8. 8.com/locate/trac .42. 7.42 ± 0.32 ± 0.65 CE CE/MS (negative detection mode) Calculated (SPARC) [30] [19] [22] N CH 3 Lomefloxacin 5.10.94 ± 0. 6. 3.04 ± 0.63 8.95 6.60 ± 0.35 ± 0.10 ± 0.01 6. 8.03 6.03 8.46.05 6.69 ± 0. 10.05. 8.56 ± 0.57 Flumequine O F COOH 6.25 ± 0.22. 5.08 Potentiometry CE CE-DAD Potentiometry CE Potentiometry/fluorimetry Potentiometry (ACN-water) Calculated (SPARC) [31] [8] [8] [3] [30] [39] [40] [22] 1050 http://www.05 6.07 8.01. 8.36 ± 0.03.11 ± 0.04.30 6. 9.20 5.30. 6.56.05. No.47 CE Calculated (SPARC) [30] [22] O F N N F N COOH Marbofloxacin O F N N O N N COOH 5.05 6.50 Potentiometry CE CE-DAD CE Calculated (SPARC) [31] [8] [8] [30] [22] N N Norfloxacine O F N HN N CH2CH3 COOH 6.00 ± 0.51 ± 0.38 ± 0.42 6.01 ± 0. 8. 6. 11.01 6.38 8.

06. No.59 ± 0.08 6.01.81 ±0. 6. pKa values can be determined from the absorbance spectra obtained at the maxima of chromatographic or electrophoretic peaks [21]. 8. SPARC [22] and ACD/Lab [23]).18 ± 0. [39] [22] O F N N N COOH Pipemidic acid O N N N N N COOH 5.97 ± 0.02 UV/pH titration (MDM-water) UV/pH titration (MeOH-water) Potentiomtery CE Potentiometry/fluorimetry Calculated (SPARC) [13] [13] [31] [30] [39] [22] Sarafloxacin O F COOH 5.Trends in Analytical Chemistry. 8.68. Vol. 8. 8. 6.18 ± 0.28 ± 0. 26.33 ± 0. 8.19 ± 0. 6.08.15 ± 0.87 ± 0.67.20 ± 0.g. That way. 2.42 ± 0. Dissociation constants are also easy to determine by the LC-MS technique.49.12.20 ± 0.  capacity factor/pH data and spectra/pH data in LC procedures (LC-DAD) [6].05.30 CE CE-DAD CE Calculated (SPARC) [8] [8] [29] [22] Ofloxacin O F N N O N COOH 6. pKa values may be obtained from two independent methods:  mobility/pH data and spectra/pH data in CE procedures (CE-DAD). The advantages of these proposed methods lie in the fact that the application of both methods does not increase the analysis time and enables the confirmation of results [5].elsevier.20 ± 0.03.04.02 8.com/locate/trac 1051 . http://www. 8.62 ± 0..12. 8.22 ± 0.11 5. 11.08.90 ± 0.02.7.88 ± 0.06 6.54 CE CE-DAD Calculated (SPARC) [8] [8] [22] N N N F (DAD) for absorbance measurements.15 ± 0. 5. 6. Computational methods Theoretical pKa values can be calculated by computational methods (e. 2007 Trends Table 3 (continued) Compound Pefloxacin Chemical structure pKa 6.05. 7.06. and.05.10 6.92 7. 7. 6. 7.09 5.25 ± 0. In these cases.09 5.21 ± 0.08 8.04 6.01 5.16.87 ± 0.83 Method Potentiometry/fluorimetry Calculated (SPARC) Ref. 8. thereby ensuring that impurities that might interfere with compounds are not detected [19]. The use of CE-MS provides high sensitivity as well as high selectivity.

elsevier.34 Method Calculated (SPARC) Ref. Dissociation constants of macrolides Compound Azithromycin H3C OH H3C OH H3C O CH3 O CH3 H O OH CH3 H O Chemical structure H3C N CH3 OH CH3 HO H3C N CH3 pKa 7. [22] O O CH3 O CH3 CH3 Clarithromycin H3C H3C HO H3C O O CH3 O OH H3C O O O CH3 O CH3 H3C OH O CH3 O CH3 CH3 CH3 HO H3C N CH3 7.15 7. 11.com/locate/trac . 2007 Table 4. Vol. No.90 ± 0.Trends Trends in Analytical Chemistry. 26.24 Potentiometry Calculated (SPARC) [3] [22] 1052 http://www.25 Calculated (SPARC) [22] Erythromycin H3C OH H3C O CH3 OH OH H3C O CH3 O O O CH3 O O H3C OH CH3 CH3 O CH3 CH3 HO H3C N CH3 8.

30 Method Potentiometry Ref. as well as specific solute-solvent interactions (solvation effects).. [3] O Tylosin 3.1. 7. dioxane and acetonitrile. several experiments at different organic solvent concentrations should be performed. The application of MDM-water mixtures improves the solubility of poorly water-soluble APIs.31 ± 0. pKa determination in non-aqueous media For substances sparingly soluble in water. multi-component. the specific solute-solvent interactions should not be too different from those established in water. 11. the Yasuda-Shedolovsky method) can be used to deduce the pKa values at zero organic solvent composition [6. Measurements are performed using IUPAC standardization rules.50 ± 0. 3. Aqueous pKa values that estimate by an extrapolation procedure (e. 2007 Trends Table 4 (continued) Compound Roxithromycin N H3C HO H3C H3C OH H3C O O O CH3 O O CH3 CH3 H3C OH O CH3 CH3 OH HO CH3 H3C N CH3 Chemical structure O O O CH3 pKa 9. ACD/pKa DB is a software program that calculates accurate acid-base ionization constants (pKa values) under 25°C and zero ionic strength in aqueous solutions for almost any organic structure. Sulfonamides. http://www.24].com/locate/trac 1053 . so their pKa values can be measured in a lower proportion of organic solvent [13]. The use of water-organic mixtures requires the correct measurement of pH in these media.1.elsevier. The selected organic solvents are usually alcohols because they are amphiprotic and neutral solvents. and they show great ability to dissolve ionizable compounds. has been found to be efficient for pKa measurement of various APIs.30. not all compounds dissolve in a singlecomponent organic solvent-water mixture. comprising equal volumes of methanol.13 Potentiometry [3] SPARC is an on-line calculator that estimates the macroscopic and microscopic pKa of any organic compound solely from its chemical structure. Antibiotics 4. 26. The dissociation of uncharged substances in aqueous-organic mixtures is ruled by electrostatic interactions. so recently a new. Then.1.13].g.17 ± 0. In order to estimate aqueous pKa values. aqueous-organic solvent mixtures are used to estimate their pKa values [13. co-solvent mixture. 4. Vol. No. However. A sulfonamide contains one basic amine group (–NH2) and one acidic amide group (–NH–). 4.Trends in Analytical Chemistry. pKa values of active pharmaceutical ingredients The aqueous pKa values of APIs determined in water or extrapolated from dissociation constants determined in water-organic media are summarized in Tables 1–8. and referred to as MDM.

Quinolones. Since the discovery of nalidixic acid (the prototype antibacterial quinolone) in 1962. 26. 2). eight-membered heterocyclic aromatic compounds with a ketone group at position 4 and a carboxylic group at position 3. Tetracyclines show three pKa values of approximately 3. several structural modifications have enhanced their biological and pharmacological activities.com/locate/trac that allows hydrogen bonding between N4 and OH12a.3. and a hydrogen-bond interaction occurs with O3 [27] (see Fig. Vol. Under alkaline conditions. As indicated by their acid-dissociation constants.26].25. 6.Trends Trends in Analytical Chemistry. 6.1. 7. but analogues have additional nitrogens at position 2 (cinnolines).1. 11.39. 6. These modifications include the introduction of alkyl or aryl groups at position 1 and fluoro and piperazinyl .41. [22] O O N HO HN O NH2 S CH3 OH CH3 3.39.42 Calculated (SPARC) [22] The amine group is able to gain a proton. protonated amino group of sulfonamide and its electrically neutral conjugate base.50. No. tetracyclines contain localized charges across all pH values and only achieve an overall neutral state as zwitterions in the approximate range of pH 3–9. 4.01 2.2.01.55 ± 0.71. 9.94 3. Dissociation constants of b-lactams Compound Amoxicillin Chemical structure pKa Method Calculated (SPARC) Ref. Under neutral and acidic conditions. the N4 position becomes protonated. 1. The main nucleus usually contains one nitrogen atom (quinolines).04 2. Tetracyclines. position 8 (naphthyridines) or positions 6 and 8 (pyridopyrimidines).14 ± 0.61 Potentiometric CE-MS (positive detection mode) CE-MS (negative detection mode) Calculated (SPARC) [13] [19] [19] [22] Penicillin G (benzylpenicillin) O O N HN O S OH CH3 CH3 3. The first dissociation constant is associated with the deprotonation of C3 hydroxyl. 4. As shown in Fig. 7 and 9. 7. although the exact assignment of these dissociation constants remains controversial [3].41 Ampicillin O O N HN O NH2 S OH CH3 CH3 2. disrupting the previous conformation. Quinolones are nitrogen-containing. Ka1 is the dissociation constant for equilibrium between the positively charged.elsevier. The loss of protons from O12 and dimethylammonium constitutes Ka2 and Ka3. while the amide group is able to release proton under specific pH conditions. tetracyclines assume a conformation 1054 http://www. 3. whereas Ka2 refers to an equilibrium involving the loss of the sulfonamide proton to yield its negatively charged conjugate [3. 2007 Table 5. The basic structure of quinolones is shown in Fig.

3 ± 0. Dissociation constants of b-blockers Compound Acebutolol O H3C CH3 N H O O CH3 Chemical structure OH H N pKa 9.48 11.elsevier.21 CH3 CH3 pKa. 26.07 CE Calculated (SPARC) [18] [22] Omeprazole N S O CH3 O N CH3 H3C O CH3 4.64 9.1 9.14.06 CE Calculated (SPARC) [18] [22] Atenolol CH3 H3C N H O OH O NH2 9.1 ± 0.Trends in Analytical Chemistry.40 9.MeOH [43] 11.90 CE/MS (positive detection mode) [19] N (continued on next page) http://www.5 ± 0. [22] Alprenolol OH O H N CH3 H3C H2C 9.35 11.09 Method Calculated (SPARC) Ref. 11.38 ± 0.42 ± 0.44 ± 0.01 9.87 11.1 ± 0.1 9.48 ± 0.com/locate/trac 1055 . Vol.41 7. 2007 Trends Table 6.06 HO O NH2 Metoprolol O H3C O OH H N CH3 CH3 9.1 ± 0.35 8.07 Potentiometry (MDM-water) CE CE CE-MS (positive detection mode) Potentiometry Calculated (SPARC) Potentiometry Calculated (SPARC) [13] [17] [18] [19] [34] [22] [30] [22] Labetalol OH H N CH3 10. No.61 9. 8.1 9.

4 ± 0.06 CE CE-MS (positive detection mode) Calculated (SPARC) [17.3 ± 0.Trends Trends in Analytical Chemistry.07 Calculated (SPARC) [22] Propranolol OH O H N CH3 CH3 9.1 ± 0.07 Potentiometry Calculated (SPARC) [34] [22] N S N N H3C O HO S N H CH3 CH3 1056 http://www. 2007 Table 6 (continued) Compound Oxprenolol Chemical structure pKa pKa.2 ± 0.89 11.16 CH3 CH3 CH2 Pindolol 9.01 11.MeOH [43] 11.18] [19] [22] Sotalol OH O H3C S O N H 9.16 11.48 11.34 8.3 ± 0. 11. [34] [22] OH O O H N 9.02 Method Potentiometry Calculated (SPARC) Ref.62 9.com/locate/trac . 26.2 ± 0.elsevier.2 9.04 Calculated (SPARC) [22] OH H N O H N CH3 CH3 Practolol O H3C N H O OH 9.06 Calculated (SPARC) [22] H N CH3 CH3 Timolol O 9. No. Vol.57 9.41 H N CH3 CH3 11.49 ± 0.

45 Potentiometry (MDM-water) Potentiometry (methanol-water) CE Potentiometry (isopropyl alcohol-water) LC Multiwavelength spectrophotometry UV/pH CE-MS (negative detection mode) Calculated (SPARC) [13] [13] [17] [24] [44] [37] [37] [19] [22] (continued on next page) http://www.21 4.30 4.16 4.24 ± 0.64 4.08 4.61 Potentiometry (isopropyl alcohol-water) LC Calculated (SPARC) [24] [44] [22] O Flurbiprofen OH O H3C F 4.40 4.63 4.52 4.56 4. Vol.com/locate/trac 1057 .45 ± 0.45 ± 0.25 Potentiometry (isopropyl alcohol-water) Calculated (SPARC) [24] [22] Cl HO Diclofenac Cl H N O HO Cl 4. 26.05 4. 2007 Trends Table 7.04 4.06 Potentiometry (isopropyl alcohol-water) CE-MS (negative detection mode) LC Calculated (SPARC) [24] [19] [44] [22] Fenbufen OH O 4.51 4. [24] [44] [22] CH3 H3C CH3 HO O 4.43 4.elsevier.03 4.42 ± 0.20 Potentiometry (isopropyl alcohol-water) LC Calculated (SPARC) [24] [44] [22] Ibuprofen CH3 H3C CH3 HO O 4.21 4.41 4. 11. No.45 Carprofen H N CH3 O 4.03 4.35 4. Dissociation constants of anti-anflammatories Compound Butibufen Chemical structure pKa Method Potentiometry (isopropyl alcohol-water) LC Calculated (SPARC) Ref.Trends in Analytical Chemistry.

23 ± 0.37 4.57 4.16 ± 0. in .76 ± 0.08.76 ± 0.70 Procaine NH2 2.71 3. 11. Vol.com/locate/trac The carboxylic group at position 3 makes these compounds acidic.28 ± 0.02 ± 0.23 ± 0.01 8. 6. respectively.25 Method Potentiometry (isopropyl alcohol-water) Calculated (SPARC) Ref.30.30. [24] [22] O OH O H3C Naproxen CH3 O OH O CH3 4.38 Potentiometry (MDM-water) Potentiometry (methanol-water) Potentiometry (isopropyl alcohol-water) LC Calculated (SPARC) [13] [13] [24] [44] [22] Table 8.38 ± 0.73 9.12 9. No. 9.84 UV/pH titration CE Calculated (SPARC) [13] [38] [22] COOCH2CH2N(C2H5)2 Trimethoprim NH2 N H2N N O CH3 CH3 O O CH3 3. The fluoro group at position 6 originates fluoroquinolones [5].09. so.84 2.03 4. 8.32 Potentiometry Calculated (SPARC) [3] [22] substitutions at positions 6 and 7.36 4. [30] [40] [3] [22] NH HN 5.Trends Trends in Analytical Chemistry. 9.33. Dissociation constants of other active pharmaceutical ingredients Compound Piperazine Chemical structure pKa Method CE Potentiometry (acetonitrile-water) Potentiometry Calculated (SPARC) Ref. 6.24. In addition.elsevier. 1058 http://www. 2007 Table 7 (continued) Compound Ketoprofen Chemical structure pKa 4.02 4. which are basic. the 7-piperazinyl quinolones include additional amine groups. 6. 26.12 4.

The reported values of pKa for acidic quinolones (flumequin and naldixic acid) range from 5.95 to 6.2 NHR SO2 NR + H2 A HA + + H H+ + A Figure 1.35 [28. In reference papers.1 NH2 NH2 + SO2 NHR SO2 H + Ka. Quinolones with only one pKa value are referred to as acidic quinolones and those with two pKa values (due to the presence of a piperazinyl ring) are called piperazinyl quinolones. Basic structure of quinolones.1 N R1 Y X R2 N R3 N R O COOK a. Some authors have suggested that three protonation/deprotonation equilibria. zwitterionic and anionic. which are cationic. 7 8 9 10 H3C CH3 CH3 R1 R2 N OH 6 11 12 5 5a 12a 3 4 1 2 OH NH2 O OH O OH OH O Figure 2. The dissociation constant of quinolones have also been determined in water-acetonitrile mixtures using different methods [31. an aqueous solution. 26. No. In order to enable LC separation of Acidic quinolones O R1 R Y X R2 N R3 COOH Ka R R1 Y X R2 N R3 O COO- Piperazinyl quinolones O R1 N N+ R H Y X R2 N R3 R H N+ COOH K a. while other quinolones can only be neutral or anionic. Dissociation equilibrium of sulfonamides.com/locate/trac 1059 .29](See Fig. Acid-base equlibria for piperazinyl quinolones and acidic quinolones. instead of two. Vol. 5). http://www.2 R1 N Y X R2 N R3 O COO- Figure 4. O 5 6 4 1 8 3 2 O OH 7 N Figure 3.Trends in Analytical Chemistry. 2007 Trends + NH3 K a.32]. 7-piperazinyl quinolones show three different species. The existing equilibria for these two types of quinolones are shown in Fig.elsevier. are involved in quinolones with a piperazinyl substituent [30] while others have reported four pKa values because fluoroquinolones contain one carboxylic group and three basic nitrogen sites [3]. 11. there is no agreement on the assignment of pKa values obtained. 4. Structure of tetracyclines.

4. but that is not the case for LC. A high concentration of the drug is required ( P 10À4 M) in order to obtain a worthwhile increase in pH at the equivalence point. 4. macrolides have only one pKa value around 9.1. small errors in the preparation of the standard will be noted as systematic deviations. S. quinolones. To overcome problems of low water solubility. For poorly soluble compounds. Education and Sports Projects (125-1253008-1350 – Advanced analytical methods for pharmaceuticals determination in the environment and 125-2120898-3148 – Croatian nomenclature in analytical chemistry).31) may correspond to the tartarate moiety [3]. Chemosphere 46 (1998) 357. the retention factors (k) used to estimate the pKa are derived from the retention time of a solute and the hold-up time. References [1] B. F.1. Vol. This problem can be overcome by using a mass spectrometer as a detector.com/locate/trac Acknowledgement This work has been supported by the EU EMCO Project (INCO CT 2004-509188 – Reduction of Environmental Risks.6–9. Lanzky.Trends Trends in Analytical Chemistry.43].H. b-lactams. H. 1060 http://www.N. In LC. Concluding remarks Several advanced analytical methods have been used for the determination of dissociation constants of APIs.7). 11. Posed by Emerging Contaminants through Advanced Treatment of Municipal and Industrial wastes) and Croatian Ministry of Science. 4. 2007 CH3 O OH CH3 O N H CH3 5.C. due to their presence in the molecule of carboxylic and amino groups [33]. but the procedure will be affected by errors in the pH measurement. P. so the pKa1 value of tylosin (3. so the pKa values of b-blockers have been determined in methanol [34. so it has only one pKa value (in the range 8.F.3. S. intermediate forms (zwitterions). Additionally. so that calibration parameters are the same for all the titration data corresponding to the same experimental run. Only tylosin has a pKa2 value (about 7. CE permits pKa determination in aqueous solutions without difficulties. 26. these methods as well as CEDAD cannot offer sufficient sensitivity to measure compounds with aqueous solubilities below 10 lM. The potentiometric methodology gives the best accuracy and better precision than any of the methods mentioned.4. Jorgensen.MeOH. the pKa values of b-blockers have been determined by CE [18] combined with a short-end injection [17].E. in which retention can be very significant without addition of an organic modifier. The LC-DAD procedure does not use retention factors. Lutzhoft. Acid-base equilibrium of atenolol. Macrolides. . Tylosin is produced as tartarate. which corresponds to the dimethylamine group. Penicillins in aqueous solution are present as neutral. the pH of the mobile phase is measured against the pH of a standard. the pKa values of drugs can be determined in media only where suitable retention is obtained. This is mainly due to the fact that the electrode system is calibrated before each potentiometric titration. The drawback of this procedure is the possibility of small changes in the electrode response every time it is immersed in a different solution. so. anionic or. The major difficulty in obtaining reliable values for the dissociation constants of b-blockers by potentiometry is their low solubility in aqueous solutions.50). in the case of amoxicillin and ampicillin. 4. + N H CH3 Ka OH + +H O NH2 O NH2 Figure 5.35] or MDM [13]. Instead. according to their chemical structure. Table 6 summarizes the aqueous pKa values of b-blockers as well as pKa. Moreover. in the LC methodology. which is able to gain a proton. it uses spectra measured by a DAD. and the electrode remains in the solution during entire titration. Ingerslev. A macrolide contains a basic dimethylamine [–N(CH3)2] group. b-blockers A b-blocker contains one basic amine group (–NH2) able to gain a proton.elsevier. By comparison. Anti-inflammatories Anti-inflammatories are sparingly soluble in water. No.2. their pKa values have been determined in water-organic media [15].5. Nielsen. giving lower precision. The average difference in aqueous and methanolic dissociation constants is about 2 pKa units. Organic solvents used are mainly alcohols [13. pKa values in reference papers have been determined in mixtures of water and an organic solvent in order to get suitable solubility. Halling-Sorensen.

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