water for the determination of acidity constants of basic drugs by capillary zone electrophoresis

Journal of Chromatography A, 1123 (2006) 113–120

Background electrolytes in 50% methanol/water for the determination of acidity constants of basic drugs by capillary zone electrophoresis Vasco de Nogales, Rebeca Ruiz, Mart´ı Ros´es, Clara R`afols, Elisabeth Bosch ∗ Departament de Qu´ımica Anal´ıtica, Universitat de Barcelona, Mart´ı i Franqu`es, 1-11, 08028 Barcelona, Spain Received 16 February 2006; received in revised form 28 April 2006; accepted 4 May 2006 Available online 24 May 2006

Abstract The acidic dissociation constants of several hydrophobic drugs, amiodarone and a series of antidepressants that show a secondary or tertiary amino group, were determined in a 50% methanol/water mixture by capillary zone electrophoresis. The electrophoretic behavior of buffers prepared from sodium acetate, tris(hydroxymethyl) aminomethane hydrochloride, sodium hydrogenphosphate, ammonium chloride, ethanolamine, butilammonium chloride, and sodium borate in the hydroalcoholic solution was tested. Thus, all of them follow the Ohm’s law until about 25 kV and, therefore, they can be used without significant Joule heat dissipation at 20 kV. For the studied drugs, buffers prepared with phosphate or borate give effective mobility measurements lower than those from other buffers. The wide pKa range of the studied drugs provides a wide pH range where the protonated forms of the amino compounds coexist with hydrogenphosphate ions and where the neutral amines coexist with boric acid. The decrease of the experimental effective mobilities in these instances can be explained through the interactions between coexisting species. Therefore, phosphate and borate buffers should be avoided to determine the mobility of amines with aqueous pKa higher than 8, at least in solutions with high methanol content. Independent measurements of acidic dissociation constants of drugs validate this statement. © 2006 Elsevier B.V. All rights reserved. Keywords: Capillary zone electrophoresis; Methanol/water buffered separation solutions in CZE; pKa of basic compounds; Amiodarone; Trazodone; Trimipramine; Imipramine; Nortriptyline; Maprotiline; 1-Aminoethylenzene; Drugs

1. Introduction Amiodarone hydrochloride is a drug widely used in treatments of heart diseases because of its antianginal and antiarrhythmic properties. It shows a very hydrophobic moiety because of its aliphatic alkyl chain and aromatic rings and a hydrophilic moiety around the tertiary ammonium, which is protonated in acidic medium (Fig. 1). Amiodarone is sparingly soluble in water, about 0.7 mg/mL [1] and forms micelles with a low critical micellar concentration, 0.5 mg/mL [2]. To obtain a limpid liquid, the amiodarone crystals have to be heated in water above 60 ◦ C. This solution, of about 50 mg/mL, remains stable when cooled down to room temperature. However, this is not a true solution but a supramolecular organization of amiodarone forming a pseudosolution [1–4], which ceased to be transparent and showed a milky opalescence at room temperature when diluted at concentration close to critical micellar concentration

∗

Corresponding author. Tel.: +34 4021228; fax: +34 4021233. E-mail address: [email protected] (E. Bosch).

0021-9673/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.chroma.2006.05.008

[3]. It was shown that the ability of this pseudosolution to be diluted without opalescence was increased when it is prepared in acidic phosphate buffer. This fact suggests some molecular interactions between amiodarone and phosphate counterions to stabilize the micellar system [4]. These physical characteristics, very low aqueous solubility and micelles formation, makes difficult the determination of classical parameters, namely pKa and hydrophobicity, of amiodarone. In fact, a number of different attempts to determine its aqueous pKa are presented in literature. Thus, published values are 6.56 ± 0.06 [1] from spectrophotometric measurements and 6.09 [5] from capillary electrophoretic measurements, both made in aqueous solution; 8.7 ± 0.2 [6] and 8.73 ± 0.07 [4] from potentiometric titrations in methanol/water mixtures and further extrapolation to aqueous solution and also a value of 8.7 ± 0.5 was obtained from interfacial measurements of pH at the air/water interface and this value is given as an intrinsic acidity constant [7]. Then, among these values, the most reliable one seems to be about 8.7. Capillary zone electrophoresis (CZE) is a simple, versatile, automated and powerful separation technique widely applied

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Fig. 1. Studied compounds.

in physicochemical profiling of pharmaceuticals. It has advantages over traditional potentiometric, spectrophotometric and other methods, as it requires very small amounts of sample and, then, it is a very convenient tool for the study of compounds with impurities and/or low aqueous solubility [8]. However, a very low value was obtained for amiodarone using aqueous CZE [5]. Other attempts to use CZE failed because of the very poor aqueous solubility of the drug and it was necessary to add an organic solvent to the buffer solution. Then, values of 6.4 and 5.4 were reported for a 4/6 (v/v) of 1-propanol/water and tetrahydrofuran/water mixtures, respectively [9]. In this work, the attempt to determine the pKa of amiodarone by CZE leads us to use a 50% methanol/aqueous buffer mixture as the BGE because this is the lowest methanol content able to keep amiodarone in solution in a wide pH range. Since this is not an ordinary BGE solution, a study about the working conditions, namely heat dissipation by Joule effect and suitability of common buffers, had to be carried out. To test the result

obtained for amiodarone, a series of antidepressants that show an amino group of known pKa in this hydroorganic solution has been studied in the same way too. 2. Experimental 2.1. Chemicals All chemicals used in the preparation of buffers were of analytical reagent grade unless otherwise noted. HPLC grade methanol, sodium hydrogenphosphate, ammonium chloride, potassium chloride, sodium hydroxide and hydrochloric acid were from Merck (Darmstadt, Germany). Sodium tetraborate and tris(hydroxymethyl)aminomethane (Tris) were purchased from Sigma–Aldrich (Steinheim, Germany). Ethanolamine (ammonium chloride ethanolamine) and benzylic alcohol were from J.T. Baker (Deventer, Holland) and butylamine was from Aldrich (Milwaukee, USA). Deionized water (Milli-Q deion-

V. de Nogales et al. / J. Chromatogr. A 1123 (2006) 113–120

izer, Millipore, Bedford, MA, USA) was used to prepare the solvent mixture. Studied compounds amiodarone hydrochloride, trazodone hydrochloride, trimipramine maleate, imipramine hydrochloride, nortriptyline hydrochloride, and maprotiline hydrochloride (Fig. 1) were purchased from Sigma–Aldrich (Steinheim, Germany) and 1-aminoethylbenzene was from Merck (Darmstadt, Germany). 2.2. Instrumentation and operational conditions

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PASTM spectrophotometric accessory (Sirius, Forest Row, UK) [11,12] which consists of a pulsed deuterium lamp, a 256element photodiode array detector and a bifurcated fibre optics dip probe. By means RefinementProTM software (Sirius, Forest Row, UK), GLpkaTM and D-PASTM were controlled and spectrophotometric data were collected and analysed. The pH change per titrant addition was limited to about 0.2 pH units. About 30 spectra and pH readings were collected from each titration. The pH electrode (combination Ag/AgCl; Sirius, Forest Row, UK) was calibrated titrimetrically in the pH range 1.8–12.2 and measurements were taken directly in the ss pH scale [10]. All experiments were done in methanol/water mixture with 0.15 M KCl under argon atmosphere at 25 ± 0.5 ◦ C using standardized 0.5 M HCl and 0.5 M KOH titrants.

2.2.1. Capillary zone electrophoresis measurements The CZE experiments were carried out with a Beckman instrument P/ACE 5500 (Palo Alto, CA, USA) equipped with a diode array spectrophotometric detector. The measurements were performed at 25 ◦ C on an uncoated fused-silica capillary (47.0 cm × 50 ␮m I.D. × 375 ␮m O.D.) obtained from Polymicro Technologies (Phoenix, USA). Before first use, the 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. Between runs with the same buffer, the capillary was rinsed 1 min with 0.1 M NaOH, 3 min with water and 5 min with running buffer. When the buffer was changed, the capillary was conditioned 5 min with 0.1 M NaOH, 10 min with water and 30 min with the new running buffer. At the end of each working session, the capillary was rinsed 10 min with deionized water and dried 1 min with N2 . Standards were injected hydrodynamically at 0.5 psi for 2 s (1 psi = 6894.76 Pa). UV detection was performed at 214 nm. The applied voltage was 20 kV of positive polarity. In all the cases, mobility measurements were done by triplicate. In Joule heating study, the working voltage was from 5 to 30 kV for each buffer. pH measurements were taken with a Ross combination electrode Orion 8102 in a Crison micropH 2002 potentiometer with a precision of ±0.1 mV (±0.002 pH units). The electrode was calibrated with ordinary aqueous buffers of pH 4.01 and 7.00 (Crison 94 63 and 94 64, respectively). Therefore, pH measurements were taken in the intersolvental sw pH scale [10].

2.3.1. Capillary zone electrophoresis measurements Stock solutions of sodium acetate, tris(hydroxymethyl) aminomethane hydrochloride (Tris), sodium hydrogenphosphate, ammonium chloride, ethanolamine, butilammonium chloride, and sodium borate were used to prepare buffers to cover the pH range 5–11. 0.05 M or 0.2 M stock solutions of each aqueous buffer were prepared and adjusted by adding the required amount of 0.5 M HCl or 0.5 M NaOH. Appropriated amount of methanol was added in order to get 50% (v/v) methanol/water BGE solutions. The ionic strength was kept constant at 0.05 M after the methanol addition. For this reason, additions of KCl were necessary to prepare sodium hydrogenphosphate buffers. The measured pH value, sw pH, was taken at 25 ◦ C and converted to ss pH using the suitable δ value, 0.13 [13]. The buffers compositions and the pH range covered by each buffer are listed in Table 1. Solutes were solved at 100 ␮g mL−1 in methanol. Benzyl alcohol (200 ␮g mL−1 ) was used as EOF marker. Buffers and solutes were filtered through a 0.45 ␮m pore size nylon filter (Whatman, Maidstone, Kent, UK) and stored in the refrigerator at 4 ◦ C until their use.

2.2.2. Spectrophotometric titrations Multiwavelength spectrophotometric titrations were performed using a GLpKaTM titrator in conjunction with a D-

2.3.2. Spectrophotometric titrations Solutions were prepared with deionised water of resistivity higher than 1014  cm−1 . In each experiment, sample solutions

2.3. Procedures

Table 1 Composition of running buffers employed in CZE at ionic strength 0.05 M w pK a w

Buffer COO−

CH3 COOH/CH3 TrisH+ /Tris H2 PO4 − /HPO4 2− NH4 + /NH3 H3 BO3 /H2 BO3 − C2 H5 ONH3 + /C2 H5 ONH2 C4 H9 ONH3 + /C4 H9 ONH2 s pH and s pK refers a s s a From Eq. (5). b c

4.7 8.1 7.2 9.2 9.2 9.5 10.7

s pK a s

5.9a 7.7a 8.2b 8.8a 9.5c 9.0a 10.1a

s pH s

range

5.0–7.0 6.7–8.7 7.2–9.2 8.0–10.0 8.5–10.5 8.0–10.0 9.0–11.0

Stock solutions for buffers 0.2 M sodium acetate, 0.5 M HCl 0.2 M Tris–HCl, 0.5 M NaOH 0.05 M disodium hydrogen phosphate, 0.5 M HCl, 0.5 M KCl 0.2 M ammonium chloride, 0.5 M NaOH 0.2 M sodium tetraborate, 0.5 M HCl 0.2 M ethanolamine hydrochloride, 0.5 M NaOH 0.2 M butylamine hydrochloride, 0.5 M NaOH

pH and pKa in methanol/water (50%) mixtures. w w pKa is pKa in aqueous solution.

From Ref. [24]. Interpolate from Ref. [25].

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about 10−5 M with a methanol content between 40 and 55% (in weight) were preacidified to a reasonably low pH value (∼3.5) with 0.5 M HCl, and then titrated with 0.5 M KOH to an appropriated high pH value (∼12.2). Titrations were carried out in the presence of potassium dihydrogenphosphate (0.25 mL of a 10 mM solution) to allow sufficient data points to be collected in the unbuffered region of the titration curve. 2.4. pKa determination 2.4.1. Capillary zone electrophoresis For a protonated base (BH+ ), the effective mobility is related to the acidity constant by means of [14,15] µBH+ µe = (1) s s  1 + 10(s pH−s pKa ) where µe is the effective mobility, µBH+ the mobility of the protonated form of the drug and ss pH and ss pKa are the pH and pKa in the binary solvent, respectively. ss pKa is defined by s  s pKa

= − log

aH+ [B] s = s pKa − log γBH+ [HB+ ]

(2)

being ss pKa the thermodynamic constant (I = 0) and γBH+ the activity coefficient of the ionic species indicated in the subscript, both quantities referred to the binary solvent. From ss pKa value, the thermodynamic ss pKa can be calculated through Eq. (2) and the Debye–H¨uckel equation, Eq. (3) log γBH+ = −

Az2 I 1/2 1 + a0 BI 1/2

(3)

where A = 0.78 and a0 B = 1.73 as given for 50% methanol/water solutions [16]. 2.4.2. Spectrophotometric titrations Target factor analysis (TFA) was used to determine s pK values from the resulting multi-wavelength spectra by a s RefinementProTM software (Sirius, Forest Row, UK). This software calculates the acidity constants in concentration terms and, therefore, determined ss pKa are the thermodynamic ones since activity coefficient terms cancel for monoprotic bases [17]. 3. Results and discussion According to the amiodarone literature pKa data, preliminary assays in aqueous buffer solutions between pH 4 and 8 and I = 50 mM were performed. Attempts performed with acetate buffers led to small, wide and tailed peaks as shown in Fig. 2a and no peak can be observed at pH higher than 5. Similar behavior is pointed out by Zhang et al. [9] when phosphate buffers at pH lower than 4.4 are used. This behavior is attributed to the high hydrophobicity of amiodarone, which cannot dissolve well in the separation medium and tends to absorb onto the capillary wall producing an obstruction of the capillary. Since the presence of chloride or acetate anions decreases the solubility of amiodarone [2], the buffer concentration was reduced to I = 20 mM and the sample concentration was reduced too. Fig. 2b shows the small and thin peaks obtained under these conditions, but

Fig. 2. Electropherograms of amiodarone with various EGB solutions. (a) Acetate buffer, I = 50 mM in aqueous solution; (b) acetate buffer, I = 20 mM in aqueous solution; (c) phosphate buffer, I = 50 mM in MeOH/H2 O (40%, 50%).

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no peak appears at pH higher than 5 either. In this instance, the reproducibility of electropherograms at pH values lower than 5 is better because the capillary is not obstructed. Literature shows that the addition of an organic cosolvent improves the quality of peaks [9,18–20] and, therefore, methanol has been selected in this work since it is the organic solvent more similar to water. Using a binary solvent with a 40% of methanol, excellent peaks were obtained at pH 5 and 7 but a distorted peak, attributed again to solubility problems, appears at pH 8. Finally, using a solution with higher methanol content, it was possible to obtain a well-shaped peak even at pH 9 and, consequently, a 50% (v/v) mixture was selected as the working solvent (Fig. 2c). Common CZE experimental conditions in several aqueous buffer/methanol mixtures, such as temperature, electrophoretic mode, buffers and applied voltage, were tested by McCalley and co-workers [21] as a preliminary work to determine the ss pKa of several basic compounds in these hydroorganic media. Studied BGE solutions were prepared from tris(hydroxymethyl)aminomethane, ethanolamine and potassium acetate and the applied voltage was 10 kV in poor methanol content BGE but 20 kV in 60–70% methanol solutions. These voltages were accurately selected to avoid the Joule heat generated by the electrical current within the capillary since any generated temperature change can affect the viscosity of the BGE and the calculated ss pKa value. In our work, to ensure the effective heat dissipation in the experimental conditions, a study of the current dependence on the applied voltage was carried out [22,23]. All the aqueous buffer/methanol solutions (50% in volume) to be employed in the mobility measurements were tested. The upper threshold voltage to get suitable heat dissipation is higher than 23 kV as shown by Ohm’s law plots given in Fig. 3 and, consequently, all the measurements are made at 20 kV. Therefore, in agreement with the buffers studied by McCalley, we recommend 20 kV as working voltage for phosphate, borate, ammonia and butylamine buffers at high methanol content. The employed buffers as well as the corresponding pKa values in both, aqueous (pKa ) and 50% methanol/water (ss pKa ) solutions, are given in Table 1. The ionic strength has been kept constant at 0.05 M. Fig. 4b shows the plots of effective mobility of amiodarone versus ss pH, which have been fitted to Eq. (1). As expected, a well-shaped sigmoidal curve is obtained. However, Fig. 4b shows that the points at ss pH higher than 8.2 and buffered by phosphate deviate from the curve showing an effective mobility lower than that expected. This behavior is consistent with the reported specific interactions between phosphate ions and the protonated form of amiodarone [4], despite any chemical reaction is proposed in the available literature to describe the chemical process. Moreover, points buffered by borate show also a deviation similar to that of points buffered by phosphate. To investigate if this anomalous electrophoretic behavior is specific for amiodarone or it is a common trend for amines, a series of tricyclic antidepressants, which show a secondary or tertiary amino group, and also the 1-aminoethylbenzene have been studied in the same way. Compounds included in this set show ss pKa values that range from 6.4 to 9.8 and, therefore,

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Fig. 3. Variation of electric current with the applied potential for various EGB solutions. Buffers prepared from acetate (♦), tris (), phosphate (), ammonia (䊉), borate (), ethanolamine () and butylamine ().

they provide a wide pH range where the cationic form of analyte can be buffered by phosphate. Studied drugs are given in Fig. 1 and their mobility curves, which show analogous behavior than amiodarone for phosphate buffers, in Fig. 4a, c–g. Thus, effective mobilities measured in phosphate buffered solutions are lower than the expected ones for all the examined amines with the exception of trazodone, which shows the lowest ss pKa , 6.42 [26]. In this instance, points buffered by phosphate are in the pH range where trazodone is in its neutral form and no deviations imputable to specific interactions are noticed. However, all the other compounds show higher ss pKa (Table 1) and, in phosphate buffers, lower effective mobilities than those expected. These deviations occur in the pH range where the protonated form of the drug and phosphate ions coexist and they increase when the s pK of the drug increases. Since deviations are more evident a s at ss pH equal or higher than the ss pKa value of H2 PO4 − , 8.2, it seems that HPO4 2− is the species that mainly interact whit the protonated base because it prevails in the buffered separation solution. Measurements using BGE prepared by borate give also slightly lower mobility values than those expected for the studied drugs and 1-aminoethylbenzene. In these instances, deviations are in the ss pH range where the deprotonated form of the drug and boric acid coexist, that is to say, in the jump region of the mobility curve at ss pH equal or lower than the ss pKa of boric acid,

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Fig. 4. Variation of the effective mobility for the studied compounds with ss pH of BGE. Symbols as in Fig. 3.

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Table 2 s pK values a s

in 50% (v/v) MeOH/H2 O, obtained from CZE and potentiometry CZE

Trazodone Amiodarone Trimipramine Imipramine 1-Aminoethylbenzene Nortriptyline Maprotiline a b

Potentiometry

µRNH+

s pK  s a

0.88 0.78 1.05 1.10 1.60 1.11 1.04

6.36 8.39 8.76 8.90 9.15 9.66 10.02

± ± ± ± ± ± ±

0.01 0.02 0.03 0.03 0.02 0.03 0.03

SD

s pK a(I=0) s

s pK a(I=0) s

0.01 0.02 0.03 0.03 0.04 0.03 0.04

6.23 8.26 8.63 8.77 9.02 9.53 9.89

6.42a 8.33b 8.46a 8.78a 9.06 9.56a 9.85a

Interpolate from values of Ref. [26]. Interpolate from values of Table 3.

9.5. Thus, interactions between neutral amino group and boric acid could explain the observed behavior. Since the main purpose of this work is to validate CZE to determine properly the dissociation constant of the very hydrophobic amiodarone, its ss pKa was computed from the fit of experimental measurements to Eq. (1) excluding points buffered by phosphate and borate. From ss pKa value, the thermodynamic s pK was calculated through Eqs. (2) and (3). Other drugs a s included in this work have been studied in the same way. Results are given in Table 2. To test the obtained ss pKa for amiodarone, its spectrophotometric determination has been carried out in several methanol/water mixtures with high methanol contents and the results are given in Table 3. They show that the interpolated value at 50% of methanol is very consistent with the electrophoretic one (Table 2). This result confirms that points buffered by phosphate and borate give abnormal effective mobilities and should be neglected in the curve fit. Potentiometric ss pKa values in several methanol/water mixtures previously determined [26] provide the value at 50% by interpolation and the results are also included in Table 2. In all these instances potentiometric s pK values agree with the electrophoretic ones when measurea s ments made using phosphate and borate buffers are excluded in the calculations. Therefore, phosphate and borate buffers are not recommended for electrophoretic studies in methanol/water mixtures of amines with w w pKa higher than 8. Similar recommendations referred to aqueous CE/MS have been reported by Wan et al. [27]. Finally, the w w pKa of amiodarone has been calculated according to the relationship s s pKa

= 0.97 w w pKa − 0.33

(5)

Table 3 Spectrophotometric ss pKa values of amiodarone %MeOH (w/w)

%MeOH (v/v)

s pK a s

39.83 41.78 44.81 46.79 49.78 51.73 54.78

45.51 47.52 50.60 52.59 55.57 57.49 60.45

8.36 8.34 8.32 8.33 8.26 8.15 8.14

± ± ± ± ± ± ±

0.02 0.03 0.01 0.01 0.03 0.01 0.01

derived for amines [28]. The calculated value is 8.85, which is consistent with those previously published by Boury et al. [4], Kr¨amer et al. [6] and Ferreira et al. [7]. 4. Conclusions Results of this work validate CZE as a suitable technique to determine the acidic dissociation constant of the very hydrophobic amiodarone. The addition of an organic solvent is necessary to keep amiodarone in solution at suitable concentration for measurements of mobility in a wide pH range. Methanol has been selected in this work because it is the organic solvent closest to water and the minimum required fraction is 50% (v/v). Hydroorganic buffers prepared from sodium acetate, tris(hydroxymethyl) aminomethane hydrochloride, sodium hydrogenphosphate, ammonium chloride, ethanolamine, butilammonium chloride, and sodium borate about 0.05 M follow the Ohm’s law until about 25 kV and, therefore, they can be used without significant Joule heat dissipation at 20 kV. Buffers prepared with phosphate or borate origin effective mobility measurements for amiodarone lower than those expected according to the values obtained from solutions prepared with other buffers. The study of other amino compounds showing a wide range of ss pKa values leads to the conclusion that interactions of analyte with buffer can distort the mobility values of the protonated amine. Thus, the protonated form of the amino compounds can interact with phosphate and the neutral amino group with boric acid resulting in a decrease of the experimental effective mobility. Therefore, to determine the mobility of amines with aqueous pKa higher than 8 in hydroorganic solutions with high methanol content, phosphate and borate buffers should be avoided. Independent measurements of acidic dissociation constants validate this statement. The extrapolation of ss pKa in 50% methanol/water to aqueous solution agrees with values previously published and validates hydroorganic CE as a useful tool to determine the pKa of very hydrophobic compounds. Acknowledgements We thank the financial support from the Ministerio de Ciencia y Tecnolog´ıa of the Spanish Government and the Fondo

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