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ˇ c´ Mirta Zrnci Sandra Babic´ ˇ c´ Pavlovic´ Dragana Mutavdzi Department of Analytical Chemistry, Faculty of Chemical Engineering and Technology, University of Zagreb, Zagreb, Croatia Received October 7, 2014 Revised January 13, 2015 Accepted January 13, 2015
Research Article
Determination of thermodynamic pKa values of pharmaceuticals from five different groups using capillary electrophoresis A determination of the thermodynamic acid dissociation constants (pKa ) of 22 frequently used pharmaceuticals using capillary electrophoresis in aqueous media is presented in this work. The investigated pharmaceuticals belong to different pharmacological groups: macrolides, fluoroquinolones, sulfonamides, -lactams, tetracyclines, and other miscellaneous pharmaceuticals. The electrophoretic mobilities of the investigated analytes were monitored in a pH range from 2.00 to 10.82. The data were fitted with an appropriate mathematical model using a nonlinear regression analysis to obtain pKa values. Experimentally obtained data were well described by the mathematical model chosen for each analyte that was confirmed by r2 values higher than 0.99 for most of the investigated analytes. Extrapolations to zero ionic strength were used to determine the thermodynamic pKa values. Experimentally obtained acid dissociation constants were interpreted using structural formulae of investigated analytes and the moieties corresponding to specific pKa were identified. Keywords: Acid dissociation constants / Capillary electrophoresis / Pharmaceuticals DOI 10.1002/jssc.201401057
Additional supporting information may be found in the online version of this article at the publisher’s web-site
1 Introduction Pharmaceuticals are complex molecules with different physicochemical and biological properties and functionality. They have an important role in the treatment and prevention of disease in both humans and animals. Large amounts of different pharmaceuticals are used worldwide and, in the last decade, their sales have been continuously increasing [1, 2]. 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 [3–7].
´ Department of AnalytCorrespondence: Professor Sandra Babic, ical Chemistry, Faculty of Chemical Engineering and Technology, ´ University of Zagreb, Marulicev trg 20, 10 000 Zagreb, Croatia E-mail: [email protected] Fax: +385-1-45-97-250
Abbreviations: AMOX, amoxicillin; AMPI, ampicillin; AZI, azithromycin; CIPRO, ciprofloxacin; CLARI, clarithromycin; ClTET, chlortetracycline; ENRO, enrofloxacin; ERY, erythromycin; NOR, norfloxacin; OTC, oxytetracycline; PG, penicillin G; PIP, piperazine; PROC, procaine; ROXY, roxithromycin; SDIAZ, sulfadiazine; SGUA, sulfaguanidine; SMETH, sulfamethazine; SMETOX, sulfamethoxazole; TET, tetracycline; TYL, tylosin; TIA, tiamulin; TMP, trimethoprim C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Relevant processes determining the fate and behavior of pharmaceuticals in the environment are sorption to soils and sediments, complexation with metals and organics, chemical oxidation with natural or water-treatment oxidants, photolysis, volatilization, and biodegradation [8]. Since most pharmaceutical compounds have acidic and/or basic functionalities, their ionization state is controlled by both solution pH and acid dissociation constants. Cationic, neutral, or anionic forms of substances 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. Acid dissociation constants can be used to describe the major species of pharmaceuticals that are present in the environment, usually in the neutral pH. Besides being useful in predicting environmental fate of pharmaceuticals, the acid dissociation constant is of fundamental importance in a wide range of applications and research areas. The acid–base property of a pharmaceutical substance is one of several crucial properties used to estimate the absorption, distribution, metabolism, and excretion of compounds in biological systems [9]. Knowledge of pKa values as a function of solvent composition is also useful in optimizing LC [10, 11] or CE [12] for the separation of ionizable compounds. The chromatographic retention and electrophoretic behavior of ionizable compounds strongly depend on the pKa of the compound and the pH of a mobile phase [13, 14]. www.jss-journal.com
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Despite the importance, the pKa values of many relevant pharmaceuticals are either not known accurately or not available at all. There is limited experimental data in the literature on the thermodynamic pKa values of pharmaceuticals. There are several methods for the determination of acid dissociation constants. In all of them, a physical property of an analyte is measured as a function of the pH of a solution. Traditionally, potentiometry [8, 15] and UV–VIS absorption spectrometry [13] have been the most useful techniques for the determination of equilibrium constants, due to their accuracy and reproducibility. Main drawbacks of potentiometric techniques include the requirements to use a milligram of pure compounds and a mixture of aqueous buffers [9]. A good alternative to potentiometric titration is UV–VIS spectrophotometry because it can handle compounds with low solubility and low sample concentrations. However, a compound must contain an UV-active chromophore close enough to the site of the acid–base function in the molecule and it is assumed that the solute of interest is pure or that its impurities do not absorb in the UV–VIS range, since the spectra of impurities can overlap with those corresponding to the solutes of interest [10, 11, 13]. An alternative to above-mentioned techniques are separation based methods (e.g. LC [16] and CE [17]). The advantages of separation techniques for the determination of pKa values are numerous. They can handle lower sample concentrations, the studied samples do not need to be pure since they are both separation techniques and sample consumption is minimal. Instruments are highly automated and require little modification for high throughput of samples. Sample concentrations are not important, only mobilities are important for calculations. CE permits pKa determination in aqueous solutions without difficulties whereas that is not the case for LC, where the retention can be influenced by composition of mobile phase [17]. One of the most important disadvantages of the LC methods is that the pH of the mobile phase and, therefore, the range of pKa values that can be determined are limited by the stability of the column package. Additionally, pKa values can also be predicted by computational methods on the basis of molecular structure (e.g. ACD Labs (https://ilab.acdlabs.com/iLab2/) and ChemSpider (http://www.chemspider.com/)). After pioneering applications of CE methods (ITP and zone electrophoresis) to pKa determination three decades ago [18–20], recently published papers about applications of CE methods bring some methodological innovation (e.g. temperature and ionic strength corrections of measured effective mobilities, determination of very low or very high pKa values, pressure assisted accelerated measurement of EOF and effective mobilities, determination of pKa in nonaqueous solvent) [21–28]. This study was focused on the determination of the thermodynamic acid dissociation constants of 22 pharmaceuticals that belong to different pharmacological groups (tetracyclines, macrolides, sulfonamides, fluoroquinolones, -lactams, and other miscellaneous pharmaceuticals) by means of CE. Reviewing available literature, for some of them C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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the information related to acid–base equilibrium was not found and this study aimed to overcome this lack of data. In this work, experimentally determined thermodynamic acid dissociation constants for azithromycin, clarithromycin, sulfaguanidine, penicillin G, and tiamulin are reported for the first time.
2 Materials and methods 2.1 Pharmaceutical standards and reagents The pharmaceuticals studied were: enrofloxacin (ENRO), ciprofloxacin (CIPRO), norfloxacin (NOR), erythromycin (ERY), roxithromycin (ROXY), azithromycin (AZI), clarithromycin (CLARI), tylosin (TYL), tetracycline (TET), chlortetracycline (ClTET), oxytetracycline (OTC), amoxicillin (AMOX), ampicillin (AMPI), penicillin G (PG), trimethoprim (TMP), piperazine (PIP), procaine (PROC), tiamulin (TIA), sulfadiazine (SDIAZ), sulfamethazine (SMETH), sulfaguanidine (SGUA), and sulfamethoxazole (SMETOX). High purity (>99%) analytical standards of pharmaceuticals were obtained mostly from Veterina Animal Health (Kalinovica, Croatia) except for NOR, ROXY, ERY (Sigma–Aldrich), and TET (Riedel-de-Haen). The chemical structures of the pharmaceuticals included in this study are shown in Supporting Information Table S1. Mass concentrations of pharmaceutical standards were 0.1 mg/mL (ENRO and NOR dissolved in 0.05 M acetic acid and 1% v/v acetone) and 1 mg/L (CIPRO, TET, ClTET, OTC, PROC, PIP, AMPI, AMOX, and PG were dissolved in 1% v/v acetone; ERY, ROXY, AZI, CLARI, TYL, and TIA were dissolved in 20% v/v acetone). TMP, SDIAZ, SGUA, SMETOX, and SMETH were prepared dissolving in 1% v/v acetone at concentration of 0.4 mg/mL. Standard solutions of pharmaceuticals were stored protected from light at 4⬚C. Chemicals used for preparation of buffers were sodium dihydrogenphosphate and disodium hydrogen phosphate, sodium hydrogen carbonate, formic acid, and sodium hydroxide from Kemika (Zagreb, Croatia), except disodium hydrophosphate that was from Merck (Darmstadt, Germany). Acetone, acidic, and phosphoric acid were supplied by Kemika (Zagreb, Croatia). Analytical reagent grade chemicals were used. Deionized water (Milli-Q deionizer, Millipore, Bedford, MA, USA) was used to prepare solvent mixture. Stock solutions of 1 M formic acid, 1 M sodium hydroxide, 0.5 M sodium dihydrogenphosphate, 1 M sodium hydrogen carbonate, 0.5 M disodium hydrogen phosphate were used to prepare buffer solutions that covered pH range from 2.00 to 10.82. The buffer solutions, with constant ionic strength of 0.05 M, were prepared by mixing the appropriate amounts of the solutions shown in Table 1. Acetone (1% v/v) was used as an EOF marker. All solutions were filtered through 0.45 m pore size hydrophilic polypropylene membrane filter (PALL Life Science, Michigan, USA) before analysis. www.jss-journal.com
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J. Sep. Sci. 2015, 0, 1–8 Table 1. Composition of BGE (50 mM ionic strength)
pH range
BGE constituents
2.00–4.76 6.10–7.98 9.22–10.82
1 M HCOOH/1 M NaOH 0.5 M NaH2 PO4 /0.5 M Na2 HPO4 1 M NaHCO3 /1 M NaOH
2.2 Instrumentation and operating conditions CE experiments were carried out with a HP3D CE system (Hewlett-Packard, Waldbronn, Germany) equipped with an autosampler, automatic injector, photodiode array detector, and an air cooling unit for the capillary. The instrument control and analysis were performed with HP Chemstation software (Hewlett-Packard, Rev. A 10.02). Uncoated fused-silica capillaries (Agilent Technologies, Waldbronn, Germany) of 75 m id, 363 m od, total length 56.0 cm and effective length (to the detector) 48.0 cm were used. The capillary cassette temperature was maintained at 25⬚C with air cooling. Samples were injected hydrodynamically at 50 mbar pressure for 2.0 s. Running voltage was 15 kV of positive polarity and the resulting electric current was 20–60 A. Running voltage was chosen based on Joule heating study where working voltages were tested in the range from 5 to 30 kV for two buffers, 2.76 and 7.98. Before the first use, the fused-silica capillary was conditioned at 25⬚C as follows: 10 min with 1 M NaOH, 10 min with 0.1 M NaOH, 10 min with water and finally 30 min with the running buffer. Before analysis the capillary was rinsed with 0.1 M NaOH for 5 min, water for 5 min and with working buffer solution used in the analysis for 10 min. When the buffer was changed, the capillary needed to be conditioned for 5 min with 0.1 M NaOH, 10 min with water and 20 min with new running buffer. After each working session, the capillary was rinsed 5 min with 0.1 M NaOH, 5 min with deionized water and dried 10 min with air. The capillary was stored dry. UV detection was performed at 254 nm for acetone as marker of EOF. Analytes were detected at 214 nm (AZI, CLARI, ERY, ROXY, OTC, TIA, TET, TYL, and ClTET), at 200 nm (AMOX, AMPI, CIPRO, ENRO, NOR, PIP, PG, SGUA, SMETH, SMETOX, and TMP), at 254 nm (SDIAZ), and at 280 nm (PROC).
2.3 Determination of effective electrophoretic mobilities The determination of pKa values by CE is based on monitoring of the effective mobility of ionized investigated substance in a series of solutions with different pH and constant ionic strength. Effective mobility, eff is calculated by: eff =
L LD V
1 1 − tS tEOF
C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
(1)
3
where tS is the migration time of the sample and tEOF is the migration time of a neutral marker compound that is of the same velocity as the EOF. LD is distance from the injection end of capillary to the detector and will be shorter than the total length of the capillary L over which the voltage V is applied. Effective mobilities were determined for pHs in the range of 2.00–10.82. Obtained data were plotted on eff –pH graphs. In all the cases, mobility measurements were done in triplicate and the % RSD values of the effective mobilities were calculated. RSDs were less than 1.5% for all the compounds.
2.4 Calculation of mixed and thermodynamic acid dissociation constants The thermodynamic acid dissociation constant of a univalent weak base can be described by the following equilibrium: BH+ + H2 O B + H3 O+
(2)
The corresponding thermodynamic dissociation constant K Ta is defined as: K aT =
aB · aH3 O+ aBH+
(3)
where aB is the activity of neutral form of compound, aH3 O+ is the activity of hydronium ion, and aBH+ is the activity of protonated species. Activities of species B and BH+ can be expressed as a product of their molar concentrations c B , c BH+ and corresponding activity coefficientsB ␥B and ␥BH+ : K aT =
c B · ␥B · aH3 O+ c BH+ · ␥BH+
(4)
The activity coefficient of a charged species can be estimated by the G¨untelberg approximation [17, 29]: √
log ␥ = −Az2
I √ 1+ I
(5)
where A = 0.51 for water at 25⬚C, z is the charge of ion, and I is the ionic strength (I = 0.5 c i zi2 ) . The determination of pKa values by CE is based on monitoring of the effective mobility of an ionizable compound in a series of electrolyte solutions of constant ionic strength and various pH values. In its uncharged state, a solute has zero mobility while its fully ionized state has maximum mobility. Intermediate mobility is therefore a function of dissociation equilibrium, so, for a univalent base, the effective mobility is given by: eff = x · ep
(6)
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where ep is the electrophoretic mobility of a cation and x is the molar fraction of the base present as the cationic form: x=
c BH+ c BH+ + c B
c B · aH3 O+ c BH+
(8)
Using Eqs. (6), (7), and (8) a model equation for K amix determination using CE for univalent base is obtained: eff =
ep · 10−pH −pK amix −pH
10
+ 10
(9)
Based on Eqs. (3) and (8), and with respect to unity value of ␥B , the relation between the thermodynamic and mixed dissociation constants can be expressed as: p K aT = p K amix + log ␥BH+
(10)
Model equations for the determination of mixed and thermodynamic pKa values for polyvalent acids and bases as well as amphoteric compounds could be derived on the same way. In cases when more than one ionization equilibrium is involved, the effective mobility is given by: eff =
Pharmaceuticals
pKa1
pKa2
pKa3
Macrolides Azithromycin Clarithromycin Erythromycin Roxithromycin Tylosin
8.96 ± 0.04 8.79 ± 0.06 8.83 ± 0.05 8.82 ± 0.04 7.71 ± 0.03
— — — — —
— — — — —
Fluoroquinolones Ciprofloxacin Enrofloxacin Norfloxacin
6.08 ± 0.08 5.70 ± 0.16 6.18 ± 0.12
Sulfonamides Sulfadiazine Sulfaguanidine Sulfamethazine Sulfamethoxazole
2.69 ± 1.97 2.21 ± 0.07 1.98 ± 0.51 (1.86 ± 1.01)
β-lactams Amoxicillin Ampicillin Penicillin G
3.20 ± 0.26 3.07 ± 0.78 2.37 ± 0.22
7.34 ± 0.05 7.12 ± 0.08 4.75 ± 0.09
10.47 ± 0.11 (11.99 ± 0.87) —
Tetracyclines Chlortetracycline Oxytetracycline Tetracycline
3.49 ± 0.24 3.71 ± 0.13 3.56 ± 0.13
7.14 ± 0.05 8.08 ± 0.19 7.09 ± 0.28
9.28 ± 0.05 10.15 ± 0.74 9.28 ± 0.18
Others Piperazine Procaine Tiamulin Trimethoprim
5.35 ± 0.11 2.17 ± 0.31 8.90 ± 0.09 7.10 ± 0.02
9.25 ± 0.08 9.95 ± 0.03 -
(7)
In the CE experiments, the pH value of the buffer is related to the activity of hydronium ion aH3 O+ , while the experimentally determined effective mobilities are related to the molar concentrations of ionic species. Therefore, “mixed” dissociation constant K amix is introduced [30]: K amix =
Table 2. Dissociation constants of investigated pharmaceuticalsa)
xi ep,i
(11)
8.41 ± 0.10 7.97 ± 0.07 8.60 ± 0.14
— — —
± ± ± ±
— — — —
6.59 (11.97 7.98 6.04
0.04 0.05) 0.03 0.04
— — — —
a) Extrapolated values are shown in parenthesis.
i
where xi is the mole fraction of the species i with its attributing electrophoretic mobility ep,i . The models for mixed and thermodynamic pKa values determination used in this work are represented in the Supporting Information Table S2. Model equations were chosen based on chemical structure of pharmaceuticals and electrophoretic mobilities of ionic species. Experimentally obtained data were well described by the mathematical model chosen that was confirmed by r2 values higher than 0.99 for most of the investigated analytes (higher than 0.98 for SGUA and ENRO).
3 Results and discussion
investigated analytes gained a proton and therefore become positively charged. Because of their ionic state they migrate before the EOF marker (acetone). Macrolide antibiotics are composed of macrocyclic lactone ring with 14, 15, or 16 carbon atoms and with one or more deoxy sugars attached to them with glycoside bond. In their structure there is a basic dimethylamine [–N(CH3 )2 ] group that is able to gain proton (Supporting Information Table S1). The thermodynamic pKa values are 8.96 ± 0.04, 8.79 ± 0.06, 8.83 ± 0.05, 8.82 ± 0.04, and 7.71 ± 0.03 for AZI, CLARI, ERY, ROXY, and TYL (Table 2), respectively, which can be attributed to dimethylamine moiety. Macrolides acid dissociation constants, experimentally proven in this paper, have not been determined for some macrolides (AZI, CLARI) or are scarce based on present literature.
3.1 Macrolides Shape of the curves showing dependence of electrophoretic mobilities on pH values for AZI, CLARI, ERY, and ROXY are similar and they have sigmoidal shape that indicates the existence of one acid dissociation constant. Electrophoretic mobility versus pH for ROXY, as an example, is shown on Fig. 1A. Electrophoretic mobilities are all positive, which shows that C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
3.2 Fluoroquinolones Analyzing mobility curves for fluoroquinolones investigated in this work (ENRO, NOR, and CIPRO) (Fig. 1B) it is not easy to observe one of the inflection points which indicates pKa . With the aid of fitting tools two pKa values can be www.jss-journal.com
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Figure 1. Electrophoretic mobilities at different pH for representative of each investigated group (A–ROXY, B–CIPRO, C–SMETH, D–AMPI, E–TET, F–PROC, G–TMP, H–TIA, I–PIP).
determined. In the structure of fluoroquinolone antibiotics there are several ionizable functional groups. They are piperazinyl group and carboxyl group and they are responsible for two acid–base equilibria that result in two possible acid dissociation constants (Supporting Information Table S1).The first ionization constant correlates to first protonization of piperazinyl group and the second constant is responsible for dissociation of carboxylic group. Fluoroquinolones have positive electrophoretic mobility up to approximately pH 7 when the positive ions are present and they are moving in the direction of EOF. In the pH range from 7 to 11, electrophoretic mobility has negative value because with deprotonation of carboxyl group negatively charged species occur. The thermodynamic pKa1 and pKa2 values of the three fluoroquinolones are located around 5.70–6.18 and 7.97–8.60, respectively (Table 2). The results obtained in this work are in good agreement with the research that report the existence of two acid dissociation constants for fluoroquinolones [31, 32] because it was considered that piperazinyl group has only one site for acid–base equilibrium. According to other research there are three acidity constants: two nitrogen atoms in the piperazinyl group and carboxylic group make totally three places in the molecule that are open to acid-base equilibrium. On the other hand it is reported the existence of four acidity constants for quinolones: carboxylic group and three basic nitrogen atoms [33].
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3.3 Sulfonamides This research showed that sulfonamides (SDIAZ, SMETH, and SMETOX) have two acid dissociation constants except for SGUA that showed only one acid dissociation constant. Sulfonamide structure contains one basic amine group (−NH2 ) and one acidic amide group (−NH−) and they correspond to pKa1 and pKa2 , respectively (Supporting Information Table S1). The amine group is able to gain a proton and therefore positively charged ionic structure occurs with positive electrophoretic migration that can be seen from the graph (Fig. 1C). When the amide group releases a proton negatively charged species is formed and its electrophoretic mobility is negative. The thermodynamic pKa values of sulfonamides are compiled in Table 2. Obtained values for three sulfonamides (SMETH, SDIAZ, and SMETOX) are in good agreement with literature data whereas for SGUA data for experimentally determined thermodynamic acid dissociation constants were not available according to the present literature overview.
3.4 -Lactams From investigated -lactam antibiotics AMOX and AMPI showed three acid dissociation constants whereas PG showed only two. Structural formulas of AMOX and AMPI reveal places in the molecule where it is possible to receive or release
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Figure 2. Presence of different ionic species under pH range from 2 to 11 (A–ROXY, B–CIPRO, C–SMETH, D–AMPI, E–TET, F–PROC, G–TMP, H–TIA, I–PIP).
a proton and therefore are the basis of explanation for acid dissociation constants. From the structural formula of penicillins carboxylic and amide groups (−NH−) can be observed as potential places for acid/base equilibrium (Supporting Information Table S1). Both of these groups at a certain pH can release a proton and the result is negatively charged molecule which travels after EOF and the values for its electrophoretic mobility are negative. PG has two acid dissociation constants and these functional groups are responsible for them. The confirmation for this assumption lies in the negative electrophoretic mobility of the PG proving the existence of negatively charged species of PG. AMOX and AMPI on the other hand have three acid dissociation constants from which two correspond to the above mentioned groups but the third is the result of amine group (Table 2). Again electrophoretic mobilities of investigated compounds can be used as confirmation for proposed dissociation pathway. Figure 1D shows positive electrophoretic mobility (at pH5) of negative specie that are a result of second and third dissociation equilibrium, i.e. the result of a proton releasing from carboxylic and amide group. Thermodynamic acid dissociation constants of three studied -lactam antibiotics are compiled in Table 2. In the literature the data on experimentally
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determined acid dissociation constants for -lactams are scarce. 3.5 Tetracyclines Investigated tetracyclines showed three acid dissociation constants. Suggested functional moieties that could be responsible for acid dissociation constants of tetracyclines are dimethylamine group, tricarbonyl group and diketone group (Supporting Information Table S1). Dimethylamine group can accept proton and therefore the molecule becomes positively charged. Positively charged species are result of first acid dissociation constant. After the second and third acidity constant molecules become negatively charged which is confirmed by negative electrophoretic mobility (Fig. 1E). Tricarbonyl and diketone groups can release a proton and therefore can be responsible for second and third acid dissociation constant. Based on data gathered in this research it is impossible to assign particular functional moieties to acid dissociation constants. Previous research assigned first acid dissociation constant to tricarbonyl group and also stated that it is not possible to assign unambiguously the pKa2 and pKa3 acid dissociation constant to the dimethylamine and diketone groups due to unsuccessive dissociation [8,34,35]. Thermodynamic acid dissociation
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constants determined in this work are in good agreement with published data (Table 2).
3.6 Others Other investigated pharmaceuticals (PROC, TMP, TIA, and PIP) showed different number and values of acid dissociation constant depending on their structural properties. PIP and PROC showed two acid dissociation constants (5.35 ± 0.11 and 9.25 ± 0.08 for PIP; 2.17 ± 0.31 and 9.95 ± 0.03 for PROC) and TIA and TMP showed only one (8.90 ± 0.09 and 7.10 ± 0.02, respectively). All these pharmaceuticals have at least one nitrogen atom in their structure, which has basic properties and therefore can accept a proton. These Molecules which are a result of this acid–base equilibrium are positively charged and this can be confirmed with the obtained results for electrophoretic mobilities that are positive for all investigated compounds (Fig. 2F–I).
3.7 Speciation of pharmaceuticals Acid dissociation constants can be used to predict the ionic species that can be found at a certain pH (Fig. 2). Speciation of pharmaceuticals is important because it enables prediction of their fate and behavior in the environment, since different species often have significantly different physicochemical properties. It is also very useful to know the behavior of investigated analyte during method development for sample preparation for LC–MS. If we use this data during method development the process can be simplified and more effective. Based on thermodynamic acid dissociation constants determined in this work, the speciation of pharmaceuticals in water is shown by their distribution curves as a function of pH value (Fig. 2).
4 Concluding remarks
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as well as with the pKa values predicted using ChemSpider and/or ACD Labs computational programs. It is well known that better accuracy and precision in pKa determination will be obtained using experimental methods, especially CE, since the choice of parameters and starting assumption could significantly affect the outcome of the calculation. However, computational methods could be very useful in the field of drug discovery where the computational methods might be used to determine physicochemical properties of the newly developed molecules without the need to synthesize all of them. Acid dissociation constants are a key figure for predicting the behavior of pharmaceuticals in the environment; they may adsorb to the sludge or sediment, undergo volatilization or remain dissolved in the water. Gathering physicochemical properties data, among others, dissociation constants, helps during risk assessment and creating prioritization lists of pharmaceuticals in the environment. This work has been supported by the Croatian Ministry of Science, Education and Sports Projects: 125-1253008-1350 Advanced analytical methods for pharmaceuticals determination in the environment. The authors have declared no conflict of interest.
5 References [1] European Medicines Agency, 2011, Trends in the sales of veterinary antimicrobial agents in nine European countries (2005–2009), (EMA/238630/2011). [2] Jjemba, P.K., Ecotoxicol. Environ. Saf. 2006, 63, 113–130. [3] Halling-Sorensen, B., Nielsen, S. N., Lanzky, P. F., Ingerslev, F., Lutzhoft, H. C. H., Jorgensen, S. E., Chemosphere 1998, 46, 357–394. [4] Daughton, C. G., Ternes T. A., Environ. Health. Perspect. 1999, 107, 907–938. ˇ M., Babic, ´ S., J. Sep. Sci. 2014, 37, 1289–1296. [5] Perisa,
On the basis of current literature data on experimentally determined acid dissociation constants are scarce or for some pharmaceutical are not available. Therefore, in this work thermodynamic acid dissociation constants for 22 pharmaceuticals from different groups (macrolides, fluoroquinolones, sulfonamides, -lactams, tetracyclines, and other miscellaneous pharmaceuticals) were determined using CE. Also good agreement of experimentally obtained data with the appropriate model were achieved that was confirmed by r2 values higher than 0.99 for most of the investigated analytes. The values for acid dissociation constants were elaborated through structural formula of investigated analytes confirming the experimental data. From these dissociation constants, the distribution of major species of pharmaceuticals as a function of pH is estimated. The obtained pKa values are in good agreement with published experimentally determined acid dissociation constants C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
´ D., Asperger, ˇ ´ D., Babic, ´ S., [6] Mutavdˇzic´ Pavlovic, D., Tolic, J. Sep. Sci. 2013, 36, 3042–3049. ´ D., Babic, ´ S., Dolar, D., Asperger, ˇ [7] Mutavdˇzic´ Pavlovic, D., ˇ c, ´ K., Horvat, A., Kastelan-Macan, ˇ Kosuti M., J. Sep. Sci. 2010, 33, 258–267. [8] Qiang, Z., Adams, C., Water Res. 2004, 38, 2874–2890. [9] Andrasi, M., Buglyo, P., Zekany, L., Gaspar, A., J. Pharm. Biomed. Anal. 2007, 44, 1040–1047. [10] Evagelou, V., Tsantili-Kakoulidou, A., Koupparis, M., J. Pharm. Biomed. Anal. 2003, 31, 1119–1128. [11] Jimenez-Lozano, E., Marques, I., Barron, D., Beltran, J.L., Barbosa, J., Anal. Chim. Acta 2002, 464, 37–45. [12] Allen, R. I., Box, K. J., Comer, J. E. A., Peake, C., Tam, K. Y., J. Pharm. Biomed. Anal. 1998, 17, 699–712. [13] Beltran, J. L., Sanli, N., Fonrodona, G., Barron, D., Ozkanb, G., Barbosa, J. Anal. Chim. Acta 2003, 484, 253– 264.
www.jss-journal.com
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ˇ c´ et al. M. Zrnci
[14] Erdemgil, F. Z., Sanli, S., Sanli, N., Ozkan, G., Barbosa, J., Guiteras, J., Beltran, J. L., Talanta 2007, 72, 489–496. [15] Wrobel, R., Chmurzynski, L., Anal. Chim. Acta 2000, 405, 303–308.
J. Sep. Sci. 2015, 0, 1–8
[26] Ehala, S., Grishina, A. A., Sheshenev, A. E., Lyapkalo, I. M., Kasicka, V., J. Chromatogr. A 2010, 1217, 8048– 8053.
[16] Janos, P., J. Chromatogr. A 2004, 1037, 15–28.
[27] Cabot, J.M., Fuguet, E., Rafols, C., Roses, M., J. Chromatogr. A 2010, 1217, 8340–8345.
[17] Poole, S. K., Patel, S., Dehring, K., Workman, H., Poole, C. F., J. Chromatogr. A 2004, 1037, 445–454.
[28] Ehala, S., Misek, J., Stara, I.G., Stary, I., Kasicka, V., J. Sep. Sci. 2008, 31, 2686–2693.
[18] Hirokawa, T., Nishino, M., Kiso, Y., J. Chromatogr. A 1982, 252, 49–65.
[29] Stumm W., Morgan J. J., Aquatic Chemistry: An Introduction Emphasizing Chemical Equilibria in Natural Waters, Wiley-VCH, New York 1981, pp. 135–246.
[19] Kasicka, V., Vacik, J., Prusik, Z., J. Chromatogr. A 1985, 320, 33–43. [20] Beckers, J. L., Everaerts, F. M., Ackermans, M.T., J. Chromatogr. A 1991, 537, 407–428. [21] Wang, J. L., Xu, X. J., Chen, D. Y., J. Pharm. Biomed. Anal. 2014, 88, 22–26. [22] Solinova, V., Kasicka, V., Electrophoresis 2013, 34(18), 2655–2665. [23] Gravador, R. S., Fuguet, E., Rafols, C., Roses, M. Electrophoresis 2013, 34, 1203–1211.
ˇ akov ´ ´ K., Zuskova, ´ I., Kenndler, E., Gas, ˇ B., Elec[30] Vcel a, trophoresis 2004, 25, 309–317. [31] Barbosa, J., Barron, D., Jimenez-Lozano, E., J. Chromatogr. A 1999, 839, 183–192. [32] Park, H.-R., Kim, T.H., Bark, K., Eur. J. Med. Chem. 2002, 37, 443–460. [33] Lin, C.-E., Deng, Y.-J., Liao, W.-S., Sun, S.-W., Lin, W.-Y., Chen, C.-C., J. Chromatogr. A 2004, 1051, 283–290.
[24] Cabot, J. M., Fuguet, E., Rafols, C., Roses, M., J. Chromatogr. A 2013, 1279, 108–116.
[34] Stephens, C. R., Murai, K., Brunings, K. J., Woodward, R.B., J. Am. Chem. Soc. 1956, 78, 4155– 4158.
[25] Fuguet, E., Rafols, C., Roses, M., J. Chromatogr. A 2011, 1218, 3928–3934.
[35] Lesson, L. J., Krueger, J. E., Nash, R. A., Tetrahedron 1963, 18, 1155–1160.
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ˇ c´ Mirta Zrnci Sandra Babic´ ˇ c´ Pavlovic´ Dragana Mutavdzi Department of Analytical Chemistry, Faculty of Chemical Engineering and Technology, University of Zagreb, Zagreb, Croatia Received October 7, 2014 Revised January 13, 2015 Accepted January 13, 2015
Research Article
Determination of thermodynamic pKa values of pharmaceuticals from five different groups using capillary electrophoresis A determination of the thermodynamic acid dissociation constants (pKa ) of 22 frequently used pharmaceuticals using capillary electrophoresis in aqueous media is presented in this work. The investigated pharmaceuticals belong to different pharmacological groups: macrolides, fluoroquinolones, sulfonamides, -lactams, tetracyclines, and other miscellaneous pharmaceuticals. The electrophoretic mobilities of the investigated analytes were monitored in a pH range from 2.00 to 10.82. The data were fitted with an appropriate mathematical model using a nonlinear regression analysis to obtain pKa values. Experimentally obtained data were well described by the mathematical model chosen for each analyte that was confirmed by r2 values higher than 0.99 for most of the investigated analytes. Extrapolations to zero ionic strength were used to determine the thermodynamic pKa values. Experimentally obtained acid dissociation constants were interpreted using structural formulae of investigated analytes and the moieties corresponding to specific pKa were identified. Keywords: Acid dissociation constants / Capillary electrophoresis / Pharmaceuticals DOI 10.1002/jssc.201401057
Additional supporting information may be found in the online version of this article at the publisher’s web-site
1 Introduction Pharmaceuticals are complex molecules with different physicochemical and biological properties and functionality. They have an important role in the treatment and prevention of disease in both humans and animals. Large amounts of different pharmaceuticals are used worldwide and, in the last decade, their sales have been continuously increasing [1, 2]. 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 [3–7].
´ Department of AnalytCorrespondence: Professor Sandra Babic, ical Chemistry, Faculty of Chemical Engineering and Technology, ´ University of Zagreb, Marulicev trg 20, 10 000 Zagreb, Croatia E-mail: [email protected] Fax: +385-1-45-97-250
Abbreviations: AMOX, amoxicillin; AMPI, ampicillin; AZI, azithromycin; CIPRO, ciprofloxacin; CLARI, clarithromycin; ClTET, chlortetracycline; ENRO, enrofloxacin; ERY, erythromycin; NOR, norfloxacin; OTC, oxytetracycline; PG, penicillin G; PIP, piperazine; PROC, procaine; ROXY, roxithromycin; SDIAZ, sulfadiazine; SGUA, sulfaguanidine; SMETH, sulfamethazine; SMETOX, sulfamethoxazole; TET, tetracycline; TYL, tylosin; TIA, tiamulin; TMP, trimethoprim C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Relevant processes determining the fate and behavior of pharmaceuticals in the environment are sorption to soils and sediments, complexation with metals and organics, chemical oxidation with natural or water-treatment oxidants, photolysis, volatilization, and biodegradation [8]. Since most pharmaceutical compounds have acidic and/or basic functionalities, their ionization state is controlled by both solution pH and acid dissociation constants. Cationic, neutral, or anionic forms of substances 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. Acid dissociation constants can be used to describe the major species of pharmaceuticals that are present in the environment, usually in the neutral pH. Besides being useful in predicting environmental fate of pharmaceuticals, the acid dissociation constant is of fundamental importance in a wide range of applications and research areas. The acid–base property of a pharmaceutical substance is one of several crucial properties used to estimate the absorption, distribution, metabolism, and excretion of compounds in biological systems [9]. Knowledge of pKa values as a function of solvent composition is also useful in optimizing LC [10, 11] or CE [12] for the separation of ionizable compounds. The chromatographic retention and electrophoretic behavior of ionizable compounds strongly depend on the pKa of the compound and the pH of a mobile phase [13, 14]. www.jss-journal.com
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ˇ c´ et al. M. Zrnci
Despite the importance, the pKa values of many relevant pharmaceuticals are either not known accurately or not available at all. There is limited experimental data in the literature on the thermodynamic pKa values of pharmaceuticals. There are several methods for the determination of acid dissociation constants. In all of them, a physical property of an analyte is measured as a function of the pH of a solution. Traditionally, potentiometry [8, 15] and UV–VIS absorption spectrometry [13] have been the most useful techniques for the determination of equilibrium constants, due to their accuracy and reproducibility. Main drawbacks of potentiometric techniques include the requirements to use a milligram of pure compounds and a mixture of aqueous buffers [9]. A good alternative to potentiometric titration is UV–VIS spectrophotometry because it can handle compounds with low solubility and low sample concentrations. However, a compound must contain an UV-active chromophore close enough to the site of the acid–base function in the molecule and it is assumed that the solute of interest is pure or that its impurities do not absorb in the UV–VIS range, since the spectra of impurities can overlap with those corresponding to the solutes of interest [10, 11, 13]. An alternative to above-mentioned techniques are separation based methods (e.g. LC [16] and CE [17]). The advantages of separation techniques for the determination of pKa values are numerous. They can handle lower sample concentrations, the studied samples do not need to be pure since they are both separation techniques and sample consumption is minimal. Instruments are highly automated and require little modification for high throughput of samples. Sample concentrations are not important, only mobilities are important for calculations. CE permits pKa determination in aqueous solutions without difficulties whereas that is not the case for LC, where the retention can be influenced by composition of mobile phase [17]. One of the most important disadvantages of the LC methods is that the pH of the mobile phase and, therefore, the range of pKa values that can be determined are limited by the stability of the column package. Additionally, pKa values can also be predicted by computational methods on the basis of molecular structure (e.g. ACD Labs (https://ilab.acdlabs.com/iLab2/) and ChemSpider (http://www.chemspider.com/)). After pioneering applications of CE methods (ITP and zone electrophoresis) to pKa determination three decades ago [18–20], recently published papers about applications of CE methods bring some methodological innovation (e.g. temperature and ionic strength corrections of measured effective mobilities, determination of very low or very high pKa values, pressure assisted accelerated measurement of EOF and effective mobilities, determination of pKa in nonaqueous solvent) [21–28]. This study was focused on the determination of the thermodynamic acid dissociation constants of 22 pharmaceuticals that belong to different pharmacological groups (tetracyclines, macrolides, sulfonamides, fluoroquinolones, -lactams, and other miscellaneous pharmaceuticals) by means of CE. Reviewing available literature, for some of them C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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the information related to acid–base equilibrium was not found and this study aimed to overcome this lack of data. In this work, experimentally determined thermodynamic acid dissociation constants for azithromycin, clarithromycin, sulfaguanidine, penicillin G, and tiamulin are reported for the first time.
2 Materials and methods 2.1 Pharmaceutical standards and reagents The pharmaceuticals studied were: enrofloxacin (ENRO), ciprofloxacin (CIPRO), norfloxacin (NOR), erythromycin (ERY), roxithromycin (ROXY), azithromycin (AZI), clarithromycin (CLARI), tylosin (TYL), tetracycline (TET), chlortetracycline (ClTET), oxytetracycline (OTC), amoxicillin (AMOX), ampicillin (AMPI), penicillin G (PG), trimethoprim (TMP), piperazine (PIP), procaine (PROC), tiamulin (TIA), sulfadiazine (SDIAZ), sulfamethazine (SMETH), sulfaguanidine (SGUA), and sulfamethoxazole (SMETOX). High purity (>99%) analytical standards of pharmaceuticals were obtained mostly from Veterina Animal Health (Kalinovica, Croatia) except for NOR, ROXY, ERY (Sigma–Aldrich), and TET (Riedel-de-Haen). The chemical structures of the pharmaceuticals included in this study are shown in Supporting Information Table S1. Mass concentrations of pharmaceutical standards were 0.1 mg/mL (ENRO and NOR dissolved in 0.05 M acetic acid and 1% v/v acetone) and 1 mg/L (CIPRO, TET, ClTET, OTC, PROC, PIP, AMPI, AMOX, and PG were dissolved in 1% v/v acetone; ERY, ROXY, AZI, CLARI, TYL, and TIA were dissolved in 20% v/v acetone). TMP, SDIAZ, SGUA, SMETOX, and SMETH were prepared dissolving in 1% v/v acetone at concentration of 0.4 mg/mL. Standard solutions of pharmaceuticals were stored protected from light at 4⬚C. Chemicals used for preparation of buffers were sodium dihydrogenphosphate and disodium hydrogen phosphate, sodium hydrogen carbonate, formic acid, and sodium hydroxide from Kemika (Zagreb, Croatia), except disodium hydrophosphate that was from Merck (Darmstadt, Germany). Acetone, acidic, and phosphoric acid were supplied by Kemika (Zagreb, Croatia). Analytical reagent grade chemicals were used. Deionized water (Milli-Q deionizer, Millipore, Bedford, MA, USA) was used to prepare solvent mixture. Stock solutions of 1 M formic acid, 1 M sodium hydroxide, 0.5 M sodium dihydrogenphosphate, 1 M sodium hydrogen carbonate, 0.5 M disodium hydrogen phosphate were used to prepare buffer solutions that covered pH range from 2.00 to 10.82. The buffer solutions, with constant ionic strength of 0.05 M, were prepared by mixing the appropriate amounts of the solutions shown in Table 1. Acetone (1% v/v) was used as an EOF marker. All solutions were filtered through 0.45 m pore size hydrophilic polypropylene membrane filter (PALL Life Science, Michigan, USA) before analysis. www.jss-journal.com
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J. Sep. Sci. 2015, 0, 1–8 Table 1. Composition of BGE (50 mM ionic strength)
pH range
BGE constituents
2.00–4.76 6.10–7.98 9.22–10.82
1 M HCOOH/1 M NaOH 0.5 M NaH2 PO4 /0.5 M Na2 HPO4 1 M NaHCO3 /1 M NaOH
2.2 Instrumentation and operating conditions CE experiments were carried out with a HP3D CE system (Hewlett-Packard, Waldbronn, Germany) equipped with an autosampler, automatic injector, photodiode array detector, and an air cooling unit for the capillary. The instrument control and analysis were performed with HP Chemstation software (Hewlett-Packard, Rev. A 10.02). Uncoated fused-silica capillaries (Agilent Technologies, Waldbronn, Germany) of 75 m id, 363 m od, total length 56.0 cm and effective length (to the detector) 48.0 cm were used. The capillary cassette temperature was maintained at 25⬚C with air cooling. Samples were injected hydrodynamically at 50 mbar pressure for 2.0 s. Running voltage was 15 kV of positive polarity and the resulting electric current was 20–60 A. Running voltage was chosen based on Joule heating study where working voltages were tested in the range from 5 to 30 kV for two buffers, 2.76 and 7.98. Before the first use, the fused-silica capillary was conditioned at 25⬚C as follows: 10 min with 1 M NaOH, 10 min with 0.1 M NaOH, 10 min with water and finally 30 min with the running buffer. Before analysis the capillary was rinsed with 0.1 M NaOH for 5 min, water for 5 min and with working buffer solution used in the analysis for 10 min. When the buffer was changed, the capillary needed to be conditioned for 5 min with 0.1 M NaOH, 10 min with water and 20 min with new running buffer. After each working session, the capillary was rinsed 5 min with 0.1 M NaOH, 5 min with deionized water and dried 10 min with air. The capillary was stored dry. UV detection was performed at 254 nm for acetone as marker of EOF. Analytes were detected at 214 nm (AZI, CLARI, ERY, ROXY, OTC, TIA, TET, TYL, and ClTET), at 200 nm (AMOX, AMPI, CIPRO, ENRO, NOR, PIP, PG, SGUA, SMETH, SMETOX, and TMP), at 254 nm (SDIAZ), and at 280 nm (PROC).
2.3 Determination of effective electrophoretic mobilities The determination of pKa values by CE is based on monitoring of the effective mobility of ionized investigated substance in a series of solutions with different pH and constant ionic strength. Effective mobility, eff is calculated by: eff =
L LD V
1 1 − tS tEOF
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(1)
3
where tS is the migration time of the sample and tEOF is the migration time of a neutral marker compound that is of the same velocity as the EOF. LD is distance from the injection end of capillary to the detector and will be shorter than the total length of the capillary L over which the voltage V is applied. Effective mobilities were determined for pHs in the range of 2.00–10.82. Obtained data were plotted on eff –pH graphs. In all the cases, mobility measurements were done in triplicate and the % RSD values of the effective mobilities were calculated. RSDs were less than 1.5% for all the compounds.
2.4 Calculation of mixed and thermodynamic acid dissociation constants The thermodynamic acid dissociation constant of a univalent weak base can be described by the following equilibrium: BH+ + H2 O B + H3 O+
(2)
The corresponding thermodynamic dissociation constant K Ta is defined as: K aT =
aB · aH3 O+ aBH+
(3)
where aB is the activity of neutral form of compound, aH3 O+ is the activity of hydronium ion, and aBH+ is the activity of protonated species. Activities of species B and BH+ can be expressed as a product of their molar concentrations c B , c BH+ and corresponding activity coefficientsB ␥B and ␥BH+ : K aT =
c B · ␥B · aH3 O+ c BH+ · ␥BH+
(4)
The activity coefficient of a charged species can be estimated by the G¨untelberg approximation [17, 29]: √
log ␥ = −Az2
I √ 1+ I
(5)
where A = 0.51 for water at 25⬚C, z is the charge of ion, and I is the ionic strength (I = 0.5 c i zi2 ) . The determination of pKa values by CE is based on monitoring of the effective mobility of an ionizable compound in a series of electrolyte solutions of constant ionic strength and various pH values. In its uncharged state, a solute has zero mobility while its fully ionized state has maximum mobility. Intermediate mobility is therefore a function of dissociation equilibrium, so, for a univalent base, the effective mobility is given by: eff = x · ep
(6)
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where ep is the electrophoretic mobility of a cation and x is the molar fraction of the base present as the cationic form: x=
c BH+ c BH+ + c B
c B · aH3 O+ c BH+
(8)
Using Eqs. (6), (7), and (8) a model equation for K amix determination using CE for univalent base is obtained: eff =
ep · 10−pH −pK amix −pH
10
+ 10
(9)
Based on Eqs. (3) and (8), and with respect to unity value of ␥B , the relation between the thermodynamic and mixed dissociation constants can be expressed as: p K aT = p K amix + log ␥BH+
(10)
Model equations for the determination of mixed and thermodynamic pKa values for polyvalent acids and bases as well as amphoteric compounds could be derived on the same way. In cases when more than one ionization equilibrium is involved, the effective mobility is given by: eff =
Pharmaceuticals
pKa1
pKa2
pKa3
Macrolides Azithromycin Clarithromycin Erythromycin Roxithromycin Tylosin
8.96 ± 0.04 8.79 ± 0.06 8.83 ± 0.05 8.82 ± 0.04 7.71 ± 0.03
— — — — —
— — — — —
Fluoroquinolones Ciprofloxacin Enrofloxacin Norfloxacin
6.08 ± 0.08 5.70 ± 0.16 6.18 ± 0.12
Sulfonamides Sulfadiazine Sulfaguanidine Sulfamethazine Sulfamethoxazole
2.69 ± 1.97 2.21 ± 0.07 1.98 ± 0.51 (1.86 ± 1.01)
β-lactams Amoxicillin Ampicillin Penicillin G
3.20 ± 0.26 3.07 ± 0.78 2.37 ± 0.22
7.34 ± 0.05 7.12 ± 0.08 4.75 ± 0.09
10.47 ± 0.11 (11.99 ± 0.87) —
Tetracyclines Chlortetracycline Oxytetracycline Tetracycline
3.49 ± 0.24 3.71 ± 0.13 3.56 ± 0.13
7.14 ± 0.05 8.08 ± 0.19 7.09 ± 0.28
9.28 ± 0.05 10.15 ± 0.74 9.28 ± 0.18
Others Piperazine Procaine Tiamulin Trimethoprim
5.35 ± 0.11 2.17 ± 0.31 8.90 ± 0.09 7.10 ± 0.02
9.25 ± 0.08 9.95 ± 0.03 -
(7)
In the CE experiments, the pH value of the buffer is related to the activity of hydronium ion aH3 O+ , while the experimentally determined effective mobilities are related to the molar concentrations of ionic species. Therefore, “mixed” dissociation constant K amix is introduced [30]: K amix =
Table 2. Dissociation constants of investigated pharmaceuticalsa)
xi ep,i
(11)
8.41 ± 0.10 7.97 ± 0.07 8.60 ± 0.14
— — —
± ± ± ±
— — — —
6.59 (11.97 7.98 6.04
0.04 0.05) 0.03 0.04
— — — —
a) Extrapolated values are shown in parenthesis.
i
where xi is the mole fraction of the species i with its attributing electrophoretic mobility ep,i . The models for mixed and thermodynamic pKa values determination used in this work are represented in the Supporting Information Table S2. Model equations were chosen based on chemical structure of pharmaceuticals and electrophoretic mobilities of ionic species. Experimentally obtained data were well described by the mathematical model chosen that was confirmed by r2 values higher than 0.99 for most of the investigated analytes (higher than 0.98 for SGUA and ENRO).
3 Results and discussion
investigated analytes gained a proton and therefore become positively charged. Because of their ionic state they migrate before the EOF marker (acetone). Macrolide antibiotics are composed of macrocyclic lactone ring with 14, 15, or 16 carbon atoms and with one or more deoxy sugars attached to them with glycoside bond. In their structure there is a basic dimethylamine [–N(CH3 )2 ] group that is able to gain proton (Supporting Information Table S1). The thermodynamic pKa values are 8.96 ± 0.04, 8.79 ± 0.06, 8.83 ± 0.05, 8.82 ± 0.04, and 7.71 ± 0.03 for AZI, CLARI, ERY, ROXY, and TYL (Table 2), respectively, which can be attributed to dimethylamine moiety. Macrolides acid dissociation constants, experimentally proven in this paper, have not been determined for some macrolides (AZI, CLARI) or are scarce based on present literature.
3.1 Macrolides Shape of the curves showing dependence of electrophoretic mobilities on pH values for AZI, CLARI, ERY, and ROXY are similar and they have sigmoidal shape that indicates the existence of one acid dissociation constant. Electrophoretic mobility versus pH for ROXY, as an example, is shown on Fig. 1A. Electrophoretic mobilities are all positive, which shows that C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
3.2 Fluoroquinolones Analyzing mobility curves for fluoroquinolones investigated in this work (ENRO, NOR, and CIPRO) (Fig. 1B) it is not easy to observe one of the inflection points which indicates pKa . With the aid of fitting tools two pKa values can be www.jss-journal.com
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Figure 1. Electrophoretic mobilities at different pH for representative of each investigated group (A–ROXY, B–CIPRO, C–SMETH, D–AMPI, E–TET, F–PROC, G–TMP, H–TIA, I–PIP).
determined. In the structure of fluoroquinolone antibiotics there are several ionizable functional groups. They are piperazinyl group and carboxyl group and they are responsible for two acid–base equilibria that result in two possible acid dissociation constants (Supporting Information Table S1).The first ionization constant correlates to first protonization of piperazinyl group and the second constant is responsible for dissociation of carboxylic group. Fluoroquinolones have positive electrophoretic mobility up to approximately pH 7 when the positive ions are present and they are moving in the direction of EOF. In the pH range from 7 to 11, electrophoretic mobility has negative value because with deprotonation of carboxyl group negatively charged species occur. The thermodynamic pKa1 and pKa2 values of the three fluoroquinolones are located around 5.70–6.18 and 7.97–8.60, respectively (Table 2). The results obtained in this work are in good agreement with the research that report the existence of two acid dissociation constants for fluoroquinolones [31, 32] because it was considered that piperazinyl group has only one site for acid–base equilibrium. According to other research there are three acidity constants: two nitrogen atoms in the piperazinyl group and carboxylic group make totally three places in the molecule that are open to acid-base equilibrium. On the other hand it is reported the existence of four acidity constants for quinolones: carboxylic group and three basic nitrogen atoms [33].
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3.3 Sulfonamides This research showed that sulfonamides (SDIAZ, SMETH, and SMETOX) have two acid dissociation constants except for SGUA that showed only one acid dissociation constant. Sulfonamide structure contains one basic amine group (−NH2 ) and one acidic amide group (−NH−) and they correspond to pKa1 and pKa2 , respectively (Supporting Information Table S1). The amine group is able to gain a proton and therefore positively charged ionic structure occurs with positive electrophoretic migration that can be seen from the graph (Fig. 1C). When the amide group releases a proton negatively charged species is formed and its electrophoretic mobility is negative. The thermodynamic pKa values of sulfonamides are compiled in Table 2. Obtained values for three sulfonamides (SMETH, SDIAZ, and SMETOX) are in good agreement with literature data whereas for SGUA data for experimentally determined thermodynamic acid dissociation constants were not available according to the present literature overview.
3.4 -Lactams From investigated -lactam antibiotics AMOX and AMPI showed three acid dissociation constants whereas PG showed only two. Structural formulas of AMOX and AMPI reveal places in the molecule where it is possible to receive or release
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Figure 2. Presence of different ionic species under pH range from 2 to 11 (A–ROXY, B–CIPRO, C–SMETH, D–AMPI, E–TET, F–PROC, G–TMP, H–TIA, I–PIP).
a proton and therefore are the basis of explanation for acid dissociation constants. From the structural formula of penicillins carboxylic and amide groups (−NH−) can be observed as potential places for acid/base equilibrium (Supporting Information Table S1). Both of these groups at a certain pH can release a proton and the result is negatively charged molecule which travels after EOF and the values for its electrophoretic mobility are negative. PG has two acid dissociation constants and these functional groups are responsible for them. The confirmation for this assumption lies in the negative electrophoretic mobility of the PG proving the existence of negatively charged species of PG. AMOX and AMPI on the other hand have three acid dissociation constants from which two correspond to the above mentioned groups but the third is the result of amine group (Table 2). Again electrophoretic mobilities of investigated compounds can be used as confirmation for proposed dissociation pathway. Figure 1D shows positive electrophoretic mobility (at pH5) of negative specie that are a result of second and third dissociation equilibrium, i.e. the result of a proton releasing from carboxylic and amide group. Thermodynamic acid dissociation constants of three studied -lactam antibiotics are compiled in Table 2. In the literature the data on experimentally
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determined acid dissociation constants for -lactams are scarce. 3.5 Tetracyclines Investigated tetracyclines showed three acid dissociation constants. Suggested functional moieties that could be responsible for acid dissociation constants of tetracyclines are dimethylamine group, tricarbonyl group and diketone group (Supporting Information Table S1). Dimethylamine group can accept proton and therefore the molecule becomes positively charged. Positively charged species are result of first acid dissociation constant. After the second and third acidity constant molecules become negatively charged which is confirmed by negative electrophoretic mobility (Fig. 1E). Tricarbonyl and diketone groups can release a proton and therefore can be responsible for second and third acid dissociation constant. Based on data gathered in this research it is impossible to assign particular functional moieties to acid dissociation constants. Previous research assigned first acid dissociation constant to tricarbonyl group and also stated that it is not possible to assign unambiguously the pKa2 and pKa3 acid dissociation constant to the dimethylamine and diketone groups due to unsuccessive dissociation [8,34,35]. Thermodynamic acid dissociation
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constants determined in this work are in good agreement with published data (Table 2).
3.6 Others Other investigated pharmaceuticals (PROC, TMP, TIA, and PIP) showed different number and values of acid dissociation constant depending on their structural properties. PIP and PROC showed two acid dissociation constants (5.35 ± 0.11 and 9.25 ± 0.08 for PIP; 2.17 ± 0.31 and 9.95 ± 0.03 for PROC) and TIA and TMP showed only one (8.90 ± 0.09 and 7.10 ± 0.02, respectively). All these pharmaceuticals have at least one nitrogen atom in their structure, which has basic properties and therefore can accept a proton. These Molecules which are a result of this acid–base equilibrium are positively charged and this can be confirmed with the obtained results for electrophoretic mobilities that are positive for all investigated compounds (Fig. 2F–I).
3.7 Speciation of pharmaceuticals Acid dissociation constants can be used to predict the ionic species that can be found at a certain pH (Fig. 2). Speciation of pharmaceuticals is important because it enables prediction of their fate and behavior in the environment, since different species often have significantly different physicochemical properties. It is also very useful to know the behavior of investigated analyte during method development for sample preparation for LC–MS. If we use this data during method development the process can be simplified and more effective. Based on thermodynamic acid dissociation constants determined in this work, the speciation of pharmaceuticals in water is shown by their distribution curves as a function of pH value (Fig. 2).
4 Concluding remarks
7
as well as with the pKa values predicted using ChemSpider and/or ACD Labs computational programs. It is well known that better accuracy and precision in pKa determination will be obtained using experimental methods, especially CE, since the choice of parameters and starting assumption could significantly affect the outcome of the calculation. However, computational methods could be very useful in the field of drug discovery where the computational methods might be used to determine physicochemical properties of the newly developed molecules without the need to synthesize all of them. Acid dissociation constants are a key figure for predicting the behavior of pharmaceuticals in the environment; they may adsorb to the sludge or sediment, undergo volatilization or remain dissolved in the water. Gathering physicochemical properties data, among others, dissociation constants, helps during risk assessment and creating prioritization lists of pharmaceuticals in the environment. This work has been supported by the Croatian Ministry of Science, Education and Sports Projects: 125-1253008-1350 Advanced analytical methods for pharmaceuticals determination in the environment. The authors have declared no conflict of interest.
5 References [1] European Medicines Agency, 2011, Trends in the sales of veterinary antimicrobial agents in nine European countries (2005–2009), (EMA/238630/2011). [2] Jjemba, P.K., Ecotoxicol. Environ. Saf. 2006, 63, 113–130. [3] Halling-Sorensen, B., Nielsen, S. N., Lanzky, P. F., Ingerslev, F., Lutzhoft, H. C. H., Jorgensen, S. E., Chemosphere 1998, 46, 357–394. [4] Daughton, C. G., Ternes T. A., Environ. Health. Perspect. 1999, 107, 907–938. ˇ M., Babic, ´ S., J. Sep. Sci. 2014, 37, 1289–1296. [5] Perisa,
On the basis of current literature data on experimentally determined acid dissociation constants are scarce or for some pharmaceutical are not available. Therefore, in this work thermodynamic acid dissociation constants for 22 pharmaceuticals from different groups (macrolides, fluoroquinolones, sulfonamides, -lactams, tetracyclines, and other miscellaneous pharmaceuticals) were determined using CE. Also good agreement of experimentally obtained data with the appropriate model were achieved that was confirmed by r2 values higher than 0.99 for most of the investigated analytes. The values for acid dissociation constants were elaborated through structural formula of investigated analytes confirming the experimental data. From these dissociation constants, the distribution of major species of pharmaceuticals as a function of pH is estimated. The obtained pKa values are in good agreement with published experimentally determined acid dissociation constants C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
´ D., Asperger, ˇ ´ D., Babic, ´ S., [6] Mutavdˇzic´ Pavlovic, D., Tolic, J. Sep. Sci. 2013, 36, 3042–3049. ´ D., Babic, ´ S., Dolar, D., Asperger, ˇ [7] Mutavdˇzic´ Pavlovic, D., ˇ c, ´ K., Horvat, A., Kastelan-Macan, ˇ Kosuti M., J. Sep. Sci. 2010, 33, 258–267. [8] Qiang, Z., Adams, C., Water Res. 2004, 38, 2874–2890. [9] Andrasi, M., Buglyo, P., Zekany, L., Gaspar, A., J. Pharm. Biomed. Anal. 2007, 44, 1040–1047. [10] Evagelou, V., Tsantili-Kakoulidou, A., Koupparis, M., J. Pharm. Biomed. Anal. 2003, 31, 1119–1128. [11] Jimenez-Lozano, E., Marques, I., Barron, D., Beltran, J.L., Barbosa, J., Anal. Chim. Acta 2002, 464, 37–45. [12] Allen, R. I., Box, K. J., Comer, J. E. A., Peake, C., Tam, K. Y., J. Pharm. Biomed. Anal. 1998, 17, 699–712. [13] Beltran, J. L., Sanli, N., Fonrodona, G., Barron, D., Ozkanb, G., Barbosa, J. Anal. Chim. Acta 2003, 484, 253– 264.
www.jss-journal.com
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ˇ c´ et al. M. Zrnci
[14] Erdemgil, F. Z., Sanli, S., Sanli, N., Ozkan, G., Barbosa, J., Guiteras, J., Beltran, J. L., Talanta 2007, 72, 489–496. [15] Wrobel, R., Chmurzynski, L., Anal. Chim. Acta 2000, 405, 303–308.
J. Sep. Sci. 2015, 0, 1–8
[26] Ehala, S., Grishina, A. A., Sheshenev, A. E., Lyapkalo, I. M., Kasicka, V., J. Chromatogr. A 2010, 1217, 8048– 8053.
[16] Janos, P., J. Chromatogr. A 2004, 1037, 15–28.
[27] Cabot, J.M., Fuguet, E., Rafols, C., Roses, M., J. Chromatogr. A 2010, 1217, 8340–8345.
[17] Poole, S. K., Patel, S., Dehring, K., Workman, H., Poole, C. F., J. Chromatogr. A 2004, 1037, 445–454.
[28] Ehala, S., Misek, J., Stara, I.G., Stary, I., Kasicka, V., J. Sep. Sci. 2008, 31, 2686–2693.
[18] Hirokawa, T., Nishino, M., Kiso, Y., J. Chromatogr. A 1982, 252, 49–65.
[29] Stumm W., Morgan J. J., Aquatic Chemistry: An Introduction Emphasizing Chemical Equilibria in Natural Waters, Wiley-VCH, New York 1981, pp. 135–246.
[19] Kasicka, V., Vacik, J., Prusik, Z., J. Chromatogr. A 1985, 320, 33–43. [20] Beckers, J. L., Everaerts, F. M., Ackermans, M.T., J. Chromatogr. A 1991, 537, 407–428. [21] Wang, J. L., Xu, X. J., Chen, D. Y., J. Pharm. Biomed. Anal. 2014, 88, 22–26. [22] Solinova, V., Kasicka, V., Electrophoresis 2013, 34(18), 2655–2665. [23] Gravador, R. S., Fuguet, E., Rafols, C., Roses, M. Electrophoresis 2013, 34, 1203–1211.
ˇ akov ´ ´ K., Zuskova, ´ I., Kenndler, E., Gas, ˇ B., Elec[30] Vcel a, trophoresis 2004, 25, 309–317. [31] Barbosa, J., Barron, D., Jimenez-Lozano, E., J. Chromatogr. A 1999, 839, 183–192. [32] Park, H.-R., Kim, T.H., Bark, K., Eur. J. Med. Chem. 2002, 37, 443–460. [33] Lin, C.-E., Deng, Y.-J., Liao, W.-S., Sun, S.-W., Lin, W.-Y., Chen, C.-C., J. Chromatogr. A 2004, 1051, 283–290.
[24] Cabot, J. M., Fuguet, E., Rafols, C., Roses, M., J. Chromatogr. A 2013, 1279, 108–116.
[34] Stephens, C. R., Murai, K., Brunings, K. J., Woodward, R.B., J. Am. Chem. Soc. 1956, 78, 4155– 4158.
[25] Fuguet, E., Rafols, C., Roses, M., J. Chromatogr. A 2011, 1218, 3928–3934.
[35] Lesson, L. J., Krueger, J. E., Nash, R. A., Tetrahedron 1963, 18, 1155–1160.
C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.jss-journal.com
