Determination of Some Physicochemical ... - American Chemical Society

Article Cite This: J. Chem. Eng. Data XXXX, XXX, XXX−XXX

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Determination of Some Physicochemical Properties of Mebendazole with RPLC Method Kader Poturcu and Ebru Ç ubuk Demiralay*

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Chemistry Department, Süleyman Demirel University, Isparta 32260, Turkey ABSTRACT: In this study, the retention behavior of benzimidazole anthelmintic drug mebendazole (MBZ) was investigated with a liquid chromatographic method. Influence of acetonitrile (ACN) concentration present in the mobile phase on retention measurement was examined. Analyses were carried out with a Kinetex Core−Shell C8 (Phenomenex, 150 × 4.6 mm I.D., 2.6 μm) column at 25 °C. The mobile phase pH (sspH) values were measured in ACN + water mixtures. By using the retention data obtained from this chromatographic analysis, thermodynamic dissociation constant (pKa) values of the mebendazole were investigated experimentally at a constant flow rate. Experimental data were analyzed by using a nonlinear regression program. This is the first time that the pH−retention time relationships of MBZ were evaluated by the experimental method. Moreover, the pKa values at 37 °C, solubility (log Sw), and lipophilicity (log Po/w) were calculated by using Abraham’s solute descriptors. The pKa values obtained from this study complied with the literature data acquired from other methods.

1. INTRODUCTION Anthelmintic drugs containing the benzimidazole functional group are used for the treatment of helminth infections. MBZ is one of the commonly used drugs for the treatment of these infections and is widely used for the treatment of intestinal (hookworm, whipworm, etc.) infections more than 20 years.1 More recently, MBZ has been shown to possess anticancer properties in a broad range of preclinical studies across various cancer types.2 This drug is highly lipophilic (log P 2.83) with low solubility (71.3 mg/L) and undergoes extensive first-pass metabolism. It has low absolute oral bioavailability (approximately 10%) with a half-life of 2.5 to 5.5 h in patients with normal hepatic functions.3 The pKa value is a very crucial physicochemical parameter since it determines the ionization profile of a compound at different pH values and helps to predict the behavior of drugs in pharmaceutical formulations and their pharmacokinetic properties.4 The other physicochemical properties such as lipophilicity, solubility, and permeability depend on the pKa value of the drug. The fact that neutral drug molecules can easily penetrate cell walls while ionized molecules remain in plasma to be cleared by renal excretion is directly related to pKa values. The ionization degree of the drug molecules determined by the pKa value influence certain viewpoints related to cell−drug interaction, like plasma protein binding, metabolism, tissue penetration, and target protein binding.5 Formulation procedures for optimizing drug delivery also benefit from the determination of pKa. Therefore, the pKa value of a compound is a fundamental physicochemical parameter used in medicinal chemistry, organic synthesis, and food and materials sciences.6 Furthermore, knowledge of © XXXX American Chemical Society

pKa, log Po/w, and log Sw values is important for the drug development process.7 These constants constitute important data for agreement of absorption, distribution, metabolism, excretion, and toxicology (ADMET).8 Knowledge of the acid−base equilibria of this commonly used drug is very important in the analytical method development and pharmacology.9−12 Some experimental techniques such as potentiometry,13 capillary electrophoresis,14 capillary electrophoresis−mass spectrometry,15 were used to determine pKa values of MBZ and also computational methods were used to predict physicochemical properties of this drug.16−19 Reversed-phase liquid chromatography (RPLC), which is one of the most preferred chromatographic technique in the analysis of the drug molecules, is highly preferred due to its accuracy, specificity, accuracy, speed, and easy automation. The most frequently used method for pKa determination of drug candidates is RPLC coupled to a different detector such as an ultraviolet (UV) detector.20 The effect of temperature on the equilibrium of chemical systems is well-known. Calculation of temperature dependent pKa is very important. Data on pKa values of drugs at 37 °C are very limited. The determination of the pKa value at this temperature is necessary to better understand for the biological transfer mechanisms in transferring the ionizable compounds to the cell.21 The pKa value depends on the structure of the functional group of the compound. The pKa values at 25 °C and 37 °C for compounds containing acidic functional groups are Received: February 4, 2019 Accepted: March 29, 2019

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DOI: 10.1021/acs.jced.9b00131 J. Chem. Eng. Data XXXX, XXX, XXX−XXX

Journal of Chemical & Engineering Data

Article

412) connected to a pH meter ( Mettler Toledo MA 235, Switzerland). 2.2. Chemicals and Reagents. MBZ was obtained from Sigma-Aldrich (St. Louis, MO). ACN, sodium hydroxide, orthophosphoric acid, and potassium hydrogen phthalate were analytical quality and supplied by Merck (Darmstadt, Germany). Potassium hydrogen phthalate was used as the standard buffer component for the calibration of the pH electrode in ACN + water mixtures.27,28 2.3. Liquid Chromatographic Method. The effect of ACN in the binary mixtures on the retention of MBZ was studied in three different percentages. ACN concentration in mobile phases was used in ratios ranging from 35 to 45% (v/ v). The pH of the mobile phase mixture consisting of 25 mM orthophosphoric acid was adjusted with 1 M NaOH. Taking into account the pKa value of the drug, the mobile phase pH values were arranged to values between 2.25 and 5.5. Chromatographic analyses were performed at a column temperature of 25 °C and a flow rate of 0.8 mL min−1. MBZ was monitored at 240 nm. 2.4. pKa Measurements. The pKa1 values of MBZ in ACN + water mixtures were measured at 25 °C. These values were calculated by using the nonlinear regression (NLREG) program.29 2.5. Preparation of Standard Solutions. MBZ was dissolved in a water + ACN binary mixture, which was studied for determination of pKa because it is less soluble in water. The solution prepared at 100 μg mL−1 concentration was protected from sunlight and stored at 4 °C.

approximately the same, whereas for basic functionl group containing compounds the pKa value at 37 °C is less than 25 °C.21 In this study, the pKa value at 37 °C is calculated from the experimentally determined pKa at 25 °C. For this, Abraham’s solute descriptors and the experimental pKa value at 25 °C were used in eq 1.21 ΔpK a = k 0 pK a 25° C + c0 + c1∑ α2 H + c 2∑ β2 H + c3π2 + c4R 2 + c5Vx

(1)

Using calculated differences between the values (ΔpKa), the pKa value at 37 °C is estimated by eq 2.21 ΔpK a = pK a 37° C − pK a 25° C

(2)

The log Po/w is a physicochemical constant shows that partition of the drug molecules between some biological membranes. The log Po/w calculations is carried out by descriptors by Abraham and co-workers.22 log Po/w = 0.088 + 0.562E − 1.054S + 0.034A − 3.460B + 3.814V

(3)

where E is the excess molar refraction of the compound, S is the dipolarity/polarizability, and A and B are hydrogen bond acidity and basicity, respectively.23 V is the McGowan characteristic volume.23 log Sw is another physicochemical constant used in the drug development process. Sw is the molar aqueous solubility of a drug at 25 °C. To calculate this constant, an equation (eq 4) proposed by Abraham is used in this study.24

3. RESULTS AND DISCUSSION The chromatographic retention of the benzimidazole ring containing the acidic and basic ionizable groups is dependent on the mobile phase pH.30 MBZ is an amphoteric molecule (acidic/basic substituents) and exhibits pKa1 corresponding to a weak base of the N3 benzimidazole ring (Figure 1).

log Sw = 0.395 − 0.955E + 0.320S + 1.155A + 3.255B − 0.785AB − 3.330V

(4)

where the AB term represents of hydrogen-bond interactions between acidic and basic functional groups of the drug.25 This paper primarily describes the retention behavior of MBZ that has been determined using the RPLC method in the microheterogeneity region at 25 °C. In all cases, thermodynamic mobile phase pKa1 (sspKa1) values of the studied compound have been calculated through the Debye−Hückel equation despite the fact that experimental values were obtained at constant ionic strength. The aqueous pKa1 (wwpKa1) value of this compound with low water solubility was calculated by two different extrapolation methods using the thermodynamic sspKa1 values experimentally determined in the ACN + water binary mixtures. The thermodynamic wwpKa1 values of MBZ were calculated with the mole fraction (XACN) of the ACN content in the mobile phase and Yasuda− Shedlovsky equation.26 In addition to these, pKa1 at 37 °C, log Po/w, and log Sw values were calculated using Abraham’s solute descriptors.

Figure 1. Chemical structure of MBZ.

The retention time (tR) of MBZ was determined in the Kinetex C8 column based on the change in pH and ACN content in the mobile phase. This column, selected in the study, has core−shell particles. Due to the low phase ratio of columns packed with core−shell particles, the retention of analytes on these columns is lower than their retention on columns packed with conventional fully porous particles, so their elution requires lower concentrations of organic modifiers.31 Therefore, instead of working with conventional fully porous stationary phase materials, the Kinetex C8 (150 × 4.6 mm I.D, 2.6 μm) column with core−shell particles was preferred. The pKa1 value of MBZ was calculated by a nonlinear regression program using the pH−tR relationship. Thermodynamic pKa1 of the nitrogen at position 3 (N3) was determined

2. EXPERIMENTAL SECTION 2.1. Instrumentation and Apparatus. The HPLC device (Shimadzu, Japan) consisted of a pump (LC-20AD) and UV− vis detector (SPD-20A), degasser (DGU-20A3), and a column oven (CTO-20A). Liquid chromatographic determinations were performed on a Kinetex Core−Shell C8 (Phenomenex, 150 × 4.6 mm I.D, 2.6 μm) column. HPLC grade water for preparation of solutions was obtained from Millipore (Direct Q3 UV, France). The pH measurements were done with a combination glass electrode (In Lab B



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Figure 2. Dissociation equilibria of MBZ.

Figure 3. Plots of the retention factors versus the pH of the acetonitrile + water binary mixture containing (A) 35, (B) 40, and (C) 45% v/v acetonitrile.

analyses, and the relative standard deviation values are below 2%. The chromatogram demonstrates the change of tR due to the mobile phase pH is given in Figure 4. The pKa1 value of the compound was calculated using the NLREG program29 at constant column temperature, taking into account the calculated activity coefficients at each mobile phase pH. The calculated sspKa1 values in the ACN + water binary mixtures are given in Table 1. In the microheterogeneity region (0.15 ≤ XACN ≤ 0.75), acetonitrile molecule interactions lead to a smaller influence of acetonitrile molecules on retention variations. In this region, solvation by water is preferred,32 which might explain the small decrease in pKa1 values of mebendazole when the percentage of acetonitrile increases (Table 1). In these regions (XACN ≤

for MBZ. After a pH 5.5, the retention of MBZ has decreased. As the pH range of the column was not sufficient in this area where acidic behavior was observed, the study could not be continued. The pKa2 value of the compound could not be determined because the column pH range (1.5−10) was not sufficient. The ionization equilibrium reaction of amphiprotic compound MBZ is depicted in Figure 2. The sigmoidal behavior showing the change in retention time of MBZ at the mobile phase pH at each ACN concentration (35, 40, 45% v/v), namely, the microheterogeneity region, is given in Figure 3. The peak symmetry of the compound at the selected mobile phase pH is approximately equal to 1. Retention times are the average of three repetitive C



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Figure 4. Overlaid chromatograms obtained from MBZ for a mobile phase composed of acetonitrile + water binary mixture (40:60% v/v) at different mobile phase pH.

equation.26 In this method, the dielectric constant (ε) value corresponding to the percentage by volume of acetonitrile is used. The wwpKa1 value is calculated by using the linear equation obtained by plotting sspKa1 + log[H2O] data against 1/ε values. Calculated values are given in Table 2. wwpKa1 values calculated from these two approaches are compatible with each other. The differences between the values (ΔpKa) were calculated with Abraham’s solute descriptors and sspKa1 values at 25 °C. As a continuation of this determination, the sspKa1 values at 37 °C were predicted using the experimental sspKa1 values at the studied temperature and ΔpKa. Calculated data at 37 °C are given by Table 3. In Table 3, Abraham’s solute descriptor (E, A, B, S, V) values were taken from the study by Abraham and Austin.29 As acetonitrile concentration increases, the dissociate capabilities of basic compound MBZ decrease and lead to reduction in the value of pKa. The pKa1 values at 37 °C of MBZ were determined here for the first time. In this work, the calculated s spKa1 value for MBZ is fully consistent with that reported by Ràfols et al.,13 Shalaeva et al.,14 Wan et al.,15 and Chemicalize program.19 log Po/w and log Sw values were calculated by independent variables and solute descriptors (eqs 1 and 4). The descriptors used33 for the calculation of these two physicochemical parameters are given in Table 4. The applications of eqs 3 and 4 are also given in Table 4. In the study, calculated log Po/w and log Sw values for MBZ are generally fully consistent with that reported in the literature. Kasim et al.’s34 log Sw values calculated by the experimental data are not compatible with the theoretically calculated values.

Table 1. Calculated Thermodynamic pKa1 Values by NLREG Program s spKa1

values

compound

35% (v/v)

40% (v/v)

45% (v/v)

mebendazole

3.235 (0.057)a

3.160 (0.076)

3.082 (0.095)

a

Standard deviation value.

0.75), the solutes are preferentially solvated by water and variations of pKa1value are small. pKa values of water-insoluble compounds in a water medium can be calculated by different extrapolation methods using pKa values determined in binary mixtures of water + organic solvents. In this study, sspKa1 values were plotted against XACN at 25 °C. The intercept of this linear relationship is the wwpKa1 values of MBZ (Figure 5).

Figure 5. Linear relationship between mobile phase pKa1 values and mole fractions of acetonitrile.

4. CONCLUSIONS This is the first study to describe the chromatographic behavior of MBZ and sspKa1 in ACN + water mixtures. Using these thermodynamic sspKa1 values, the wwpKa1 value was calculated

One of the most common approaches used to calculate the pKa value in water is the method of the Yasuda−Shedlovsky

Table 2. Aqueous pKa1 Values Calculated from Yasuda−Shedlovsky Equation ACN% (v/v)

ε

1/ε

[H2O]

log[H2O]

s spKa1

35 40 45 0

64.070 61.951 59.818 78.330

0.0156 0.0161 0.0167 0.0128

53.566 53.289 53.011 55.509

1.729 1.727 1.724 1.744

3.235 3.160 3.082 3.624

D

s spKa1

+ log[H2O] 4.964 4.887 4.806 5.368



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Table 3. Abraham’s Solute Descriptors and Calculated pKa1 Values at 37 °C for Mebendazole %ACN (v/v)

k0

35 40 45 0

−0.026 −0.026 −0.026 −0.026

pK25 a

°C

3.235 3.160 3.082 3.624

c0

c1

A

c2

B

c3

S

c4

E

c5

Vx

ΔpKa

°C pK37 a

−0.136 −0.136 −0.136 −0.136

0.008 0.008 0.008 0.008

0.71 0.71 0.71 0.71

0.018 0.018 0.018 0.018

1.22 1.22 1.22 1.22

0.035 0.035 0.035 0.035

2.6 2.6 2.6 2.6

−0.032 −0.032 −0.032 −0.032

2.74 2.74 2.74 2.74

0.02 0.02 0.02 0.02

2.13 2.13 2.13 2.13

−0.147 −0.145 −0.143 −0.157

3.088 3.015 2.939 3.467

benzimidazole derivatives by capillary electrophoresis. J. Sep. Sci. 2009, 32, 1087−1095. (6) Thompson, P. A.; Wright, D. E.; Counsell, C. E.; Zajicek, J. Statistical analysis, trial design and duration in Alzheimer’s disease clinical trials: a review. Int. Psychogeriatr. 2012, 24, 689−697. (7) Huo, H.; Li, T.; Zhang, L. pKa determination of oxysophocarpine by reversed - phase high performance liquid chromatography. Springerplus 2013, 2, 270−275. (8) Wang, J.; Skolnik, S. Recent Advances in Physicochemical and ADMET Profiling in Drug Discovery. Chem. Biodiversity 2009, 6, 1887−1899. (9) Yılmaz Ortak, H.; Demiralay, E. C. Effect of temperature on the retention of Janus kinase 3 inhibitor in different mobile phase compositions using reversed-phase liquid chromatography. J. Pharm. Biomed. Anal. 2019, 164, 706−712. (10) Demiralay, E. C.; Alsancak, G.; Ozkan, S. A. Determination of pKa values of nonsteroidal antiinflammatory drug-oxicams by RP− HPLC and their analysis in pharmaceutical dosage forms. J. Sep. Sci. 2009, 32, 2928−2936. (11) Talay, A.; Demiralay, E. C.; Daldal, Y. D.; Ü stün, Z. Investigation of thermodynamic acidity constants of some statins with RPLC method. J. Mol. Liq. 2015, 208, 286−290. (12) Gumustas, M.; Sanlı, S.; Sanlı, N.; Ozkan, S. A. Determination of pK(a) values of some antihypertensive drugs by liquid chromatography and simultaneous assay of lercanidipine and enalapril in their binary mixtures. Talanta 2010, 82, 1528−1537. (13) Ràfols, C.; Subirats, X.; Rubio, J.; Rosés, M.; Bosch, E. Lipophilicity of amphoteric and zwitterionic compounds: A comparative study of determination methods. Talanta 2017, 162, 293−299. (14) Shalaeva, M.; Kenseth, J.; Lombardo, F.; Bastin, A. Measurement of dissociation constants (pKa values) of organic compounds by multiplexed capillary electrophoresis using aqueous and cosolvent buffers. J. Pharm. Sci. 2008, 97, 2581−2606. (15) Wan, H.; Holmén, A.G.; Wang, Y.; Lindberg, W.; Englund, M.; Någård, M.B.; Thompson, R. A. High-throughput screening of pKa values of pharmaceuticals by pressure-assisted capillary electrophoresis and mass spectrometry. Rapid Commun. Mass Spectrom. 2003, 17, 2639−2648. (16) Rankovic, Z. CNS physicochemical property space shaped by a diverse set of molecules with experimentally determined exposure in the mouse brain. J.Med. Chem. 2017, 60, 5943−5954. (17) Pallas Software Version 3.0, 2007. CompuDrug International Inc.: Grandview Drive, South San Francisco, CA 94080, USA. (18) ACD/Labs 6.00 C; ACD/Structure Elucidator, version 15.01, Advanced Chemistry Development, Inc.: Toronto, ON, Canada, www.acdlabs.com. Accessed August 21, 2018. (19) Chemicalize, 2018. https://chemicalize.com/#/calculation. Accessed December 13, 2018. (20) Babić, S.; Horvath, A. J. M.; Pavlović, D. M.; Macan, M. K. Determination of pKa values of active pharmaceutical ingredients. TrAC, Trends Anal. Chem. 2007, 26, 1043−1061. (21) Sun, N.; Avdeef, A. Biorelevant pKa (37°C) predicted from the 2D structure of the molecule and its pKa at 25°C. J. Pharm. Biomed. Anal. 2011, 56, 173−182. (22) Abraham, M. H.; Ibrahim, A.; Zissimos, A. M.; Zhao, Y. H.; Comer, J.; Reynolds, D. P. Application of hydrogen bonding calculations in property based drug design. Drug Discovery Today 2002, 7, 1056−1063.

Table 4. Comparison of Calculated and Literature Values parameter log Po/w log Sw

calculated 2.814 −4.308

literature values 34

2.260 −3.74034

2.50034 2.71035

3.26019 −4.94019

with different extrapolation methods. The prediction model was used for calculation of pKa values at 37 °C, which is scarcely reported in the literature, using experimentally determined sspKa1values at 25 °C. log Po/w and log Sw of MBZ in water were predicted with Abraham’s solute descriptors. By this study, the calculated physicochemical parameters will provide an understanding of the transport process of the drug molecules through biological systems and the advancement of nanostructures for the transport of the drug.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected], ebrucubuk@sdu. edu.tr. Tel: +90 246 2114167. Fax: +90 246 2371106. ORCID

Ebru Ç ubuk Demiralay: 0000-0002-6270-7509 Funding

The authors acknowledge the financial support of Süleyman Demirel University through the Institutional Research Fund (Project number: 5020-Ö YP-D2-17) and Ö YP project (Project number: Ö YP-05234-Dr-2013). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Dr. Jose Luis Beltran (University of Barcelona) for kindly providing the NLREG 4.0 program. REFERENCES

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(23) Abraham, M. H.; Le, J. The correlation and prediction of the solubility of compounds in water using an amended solvation energy relationship. J. Pharm. Sci. 1999, 88, 868−880. (24) Stovall, D. M.; Chelsea, G.; Keown, S.; Hoover, K. R.; Barnes, R.; Harris, C.; Lozano, J.; Nguyen, M.; Rodriguez, E.; Acree, W. E., Jr.; Abraham, M. H. Solubility of crystalline nonelectrolyte solutes in organic solvents: mathematical correlation of 4-chloro-3-nitrobenzoic acid and 2-chloro-5-nitrobenzoic acid solubilities with the Abraham solvation parameter model. Phys. Chem. Liq. 2005, 43, 351−360. (25) Abraham, M. H.; Ibrahim, A.; Zissimos, A. M. Determination of sets of solute descriptors from chromatographic measurements. J. Chromatogr. A. 2004, 1037, 29−47. (26) Yasuda, M. Dissociation Constants of Some Carboxylic Acids in Mixed Aqueous Solvents. Bull. Chem. Soc. Jpn. 1959, 32, 429−432. (27) Mussini, T.; Covington, A. K.; Longhi, P.; Rondinini, S. Criteria for Standardization of pH Measurements in Organic Solvents and Water + Organic Solvent Mixtures of Moderate to High Permittivities. Pure Appl. Chem. 1985, 57, 865−876. (28) Rondinini, S.; Mussini, P. R.; Mussini, T. Reference Value standards and Primary standards for pH Measurements in Organic Solvents and water + organic Solvent Mixtures of Moderate to High Permittivities. Pure Appl. Chem. 1987, 59, 1549−1560. (29) Sherrod, P. H. NLREG Version 4.0. http://www.nlreg.com/ (Accessed November 8, 2018). (30) Sher, N.; Fatima, N.; Perveen, S.; Siddiqui, F. A. Determination of benzimidazoles in pharmaceuticals and human serum by highperformance liquid chromatography. Instrum. Sci. Technol. 2016, 44, 672−682. (31) Guiochon, G.; Gritti, F. Shell particles, trials, tribulations and triumphs. J. Chromatogr. A. 2011, 1218, 1915−1938. (32) Barbosa, J.; Sanz-Nebot, V. Preferential solvation in acetonitrile−water mixtures. Relationship between solvatochromic parameters and standard pH values. J. Chem. Soc., Faraday Trans. 1994, 90, 3287−3292. (33) Abraham, M. H.; Austin, R. P. The effect of ionized species on microsomal binding. Eur. J. Med. Chem. 2012, 47, 202−205. (34) Kasim, N. A.; Whitehouse, M.; Ramachandran, C.; Bermejo, M.; Lennernäs, H.; Hussain, A. S.; Junginger, H. E.; Stavchansky, S. A.; Midha, K. K.; Shah, V. P.; Amidon, G. L. Molecular Properties of WHO Essential Drugs and Provisional Biopharmaceutical Classification. Mol. Pharmaceutics 2004, 1, 85−96. (35) Horvat, A. J. M.; Babić, S.; Pavlović, D.M.; Ašperger, D.; Pelko, S.; Kaštelan-Macan, M.; Petrović, M.; Mance, A.D. Analysis, Occurrence and Fate of Anthelmintics and Their Transformation Products in the Environment. TrAC Trends Analyt. Chem. 2012, 31, 61−84.

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Determination of Some Physicochemical ... - American Chemical Society

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