CHIRALITY (2014)
Chiral Separation of Four Stereoisomers of Ketoconazole Drugs Using Capillary Electrophoresis WAN AINI WAN IBRAHIM,1,2,3* SITI ROSILAH ARSAD,1,2,3 HASMERYA MAAROF,1,2,3 MOHD. MARSIN SANAGI,1,2,3 4 AND HASSAN Y. ABOUL-ENEIN ** 1 Separation Science & Technology Group (SepSTec), Department of Chemistry, Faculty of Science, Sukdai, Johor Bahru, Johor, Malaysia 2 Nanotechnology Research Alliance, Universiti Teknologi Malaysia, Sukdai, Johor Bahru, Johor, Malaysia 3 Ibnu Sina Institute for Fundamental Science Studies, Universiti Teknologi Malaysia, Sukdai, Johor Bahru, Johor, Malaysia 4 Department of Pharmaceutical and Medicinal Chemistry, Pharmaceutical and Drug Industries Research Division, National Research Centre, Dokki, Cairo, Egypt
ABSTRACT This work aimed to develop a chiral separation method of ketoconazole enantiomers using electrokinetic chromatography. The separation was achieved using heptakis (2, 3, 6tri-O-methyl)-β-cyclodextrin (TMβCD), a commonly used chiral selector (CS), as it is relatively inexpensive and has a low UV absorbance in addition to an anionic surfactant, sodium dodecyl sulfate (SDS). The influence of TMβCD concentration, phosphate buffer concentration, SDS concentration, buffer pH, and applied voltage were investigated. The optimum conditions for chiral separation of ketoconazole was achieved using 10 mM phosphate buffer at pH 2.5 containing 20 mM TMβCD, 5 mM SDS, and 1.0% (v/v) methanol with an applied voltage of 25 kV at 25 °C with a 5-s injection time (hydrodynamic injection). The four ketoconazole stereoisomers were successfully resolved for the first time within 17 min (total analysis time was 28 min including capillary conditioning). The migration time precision of this method was examined to give repeatability and reproducibility with RSDs ≤5.80% (n =3) and RSDs ≤8.88% (n =9), respectively. Chirality 00:000-000, 2014. © 2014 Wiley Periodicals, Inc. KEY WORDS: electrokinetic chromatography; chiral separation; azole drugs; ketoconazole; heptakis (2,3,6-tri-O-methyl)-β-cyclodextrin; sodium dodecyl sulfate INTRODUCTION
Chirality has been and still remains one of the major issues in drug development and the pharmaceutical industry. The United States Food and Drug Administration, European Committee for Proprietary Medicinal Products, and other drug controlling agencies have issued certain guidelines to pharmaceutical and agrochemical industries about the marketing of racemates.1 In the pharmaceutical industry, the separation of enantiomers is growing in interest because the enantiomers of a compound can display quite different activity and toxicity profiles. For this reason, the U.S. Food and Drug Administration has endorsed the development of enantiomerselective synthesis and analysis methods when a new drug is going to be developed and marketed.2 There is no doubt that enantiomer separation by capillary electrophoresis (CE) has rapidly attracted attention as a promising technique due to its high separation efficiency and flexibility. Among various different CE modes, capillary zone electrophoresis (CZE) and electrokinetic chromatography (EKC), in which only a chiral selector is added to the usual running buffer solution, are the most widely used for enantiomer separations.3,4 One of the most attractive advantages of EKC for the separation of enantiomers is an easy change of separation media in the method development; that is, one can easily alter the separation solution to find the optimum separation media and one can also use an expensive chiral selector because of the small amounts required.5 Ketoconazole was the first orally active azole introduced in clinical practice as an effective antifungal agent. Ketoconazole is chiral and has two stereogenic centers in the molecule. Their absolute configuration has been determined via synthesis by Rotstein et al.6 as cis-2R,4S and 2S,4R, also trans-2R4R © 2014 Wiley Periodicals, Inc.
and 2S4S (Fig. 1). The trans diastereomers were found to be a much weaker inhibitor than the cis pair. Earlier chiral separation of ketoconazole was carried out using supercritical fluid chromatography (SFC) and highperformance liquid chromatography (HPLC) on a chiral stationary phase based on substituted polysaccharides with different groups such as 3,5-dimethylphenylcarbamates and (R)-phenyl-ethylcarbamate. The separation of ketoconazole using SFC and HPLC only gives two stereoisomers with Rs =0.72–0.83, k =3.7–12.2 min and Rs =1.53 - 1.70, k =10.8–17.2 min, respectively.7 In the year 2000, the chiral separation of ketoconazole using SFC on an amylosebased column was described. Two stereoisomers of ketoconazole were resolved in less than 7 min with high resolution (Rs =4.29).8 Recently, the chiral separation of ketoconazole was carried out by using EKC, which focused on investigating the applicability of a chiral selector to recognize the enantiomers stereoselectivity. The most widely used chiral selector in EKC are cyclodextrins, due to their low UV absorbance, relative inexpense, ease of use, and ready availablity.9,10 Only two stereoisomers of ketoconazole were successfully resolved using EKC with TMβCD as the chiral selector in a 100 mM *Correspondence to: Wan Aini Wan Ibrahim, Separation Science & Technology Group (SepSTec), Department of Chemistry, Faculty of Science, UTM, 81310 UTM Johor Bahru, Johor, Malaysia. E-mail: [email protected] or [email protected]; Hassan Y. Aboul-Enein, Department of Pharmaceutical and Medicinal Chemistry, National Research Centre, Dokki, Cairo 12311, Egypt. E-mail: [email protected] Received for publication 13 July 2014; Accepted 27 October 2014 DOI: 10.1002/chir.22416 Published online in Wiley Online Library (wileyonlinelibrary.com).
WAN IBRAHIM ET AL.
Fig. 1. Stereoisomers of ketoconazole.
phosphate buffer (pH 3.5). Good precision was obtained, where the RSD was found to be lower than 1.2, 4.9, and 6.1% for migration time, enantiomeric resolution, and corrected peak areas, respectively.10 The use of high buffer concentration is not favorable in CE analysis, as it will generate high current and lead to Joule heating.11–13 To the best of our knowledge, only two stereoisomers of ketoconazole were successfully resolved using SFC, HPLC, and CE. Thus, in this study we modified and optimized the composition of background electrolyte (BGE), which is the key parameter in CE to separate all four stereoisomers of ketoconazole. Our main interest was to apply an anionic surfactant solution, sodium dodecyl sulfate (SDS), in the BGE, which has been the most popular technique for many years. Generally, addition of anionic surfactant solution into BGE at a higher concentration than the critical micelle concentration (CMC) is effective for the separation of ionic analytes and also electrically neutral or nonionic analytes. Thus, the addition of anionic surfactant into BGE is suitable for drug analyses involving cationic, anionic and neutral drugs.14 SDS functions as an anionic selector and also carrier for the neutral analyte ketoconazole. MATERIALS AND METHODS Chemicals and Reagents Ketoconazole was purchased from Dr Ehrenstorfer (Augsburg, Germany) (batch number C14532000, 99.0% purity), heptakis(2,3,6-tri-Omethyl)-β-cyclodextrin (TMβCD) was purchased from Sigma-Aldrich 1 (St. Louis, MO), (batch number H4645-5G, MW: 1429.54 g mol , purity ≥90%), and sodium dodecyl sulfate (SDS) was obtained from Fisher Scientific (Loughborough, UK). Sodium hydroxide (NaOH) and disodium hydrogen phosphate 12-hydrate were purchased from Riedel-de Haen (Seelze, Germany). All other chemicals and solvents were common brands of analytical-reagent grade. Water used for dilutions or as buffer preparation was produced from a Water Purification System from 1 Millipore (Molsheim, France). The stock solution (1000 mg L ) of the ketoconazole was prepared by dissolving the drug in HPLC-grade metha1 nol and diluted to 100 mg L as a typical concentration of sample to be injected. All the separation solutions were filtered through a 0.20-μm nylon syringe filter from Whatman (Clifton, NJ).
Method All electropherograms were obtained with the G1600A Agilent capillary electrophoresis system from Agilent Technologies (Waldbronn, Germany), equipped with temperature control and diode array detection (DAD). Separations were performed using an untreated fused silica capillary of 64.5 cm × 50 μm i.d. (with an effective length of 56 cm to the Chirality DOI 10.1002/chir
detector window) obtained from Polymicro Technologies (Phoenix, AZ). Background electrolytes (BGE) were prepared by dissolving anionic surfactant, SDS, and TMβCD at an adequate concentration in a solution of disodium hydrogen phosphate 12-hydrate where phosphoric acid was used to adjust the preferred pH of the phosphate buffer. Before use, the new capillary was rinsed with 1.0 M NaOH solution for 40 min, 0.1 M NaOH solution for 40 min, with deionized water for 40 min, and finally with the running buffer for 40 min. In between runs, the capillary was conditioned with 0.1 M NaOH for 3 min, followed by deionized water for 3 min and with BGE for 5 min.
RESULTS AND DISCUSSION Method Development
The method development for chiral separation was initially performed by optimizing the experimental parameters such as concentration of chiral selector, concentration of running electrolyte, pH of buffer solution, as well as the use of additives and the presence of an organic modifier. In addition, the effect of different instrumental conditions such as applied voltage and separation temperature was also investigated. In a previous article10 regarding the chiral separation of ketoconazole by EKC, we noted that the use of TMβCD as a chiral selector with a high concentration of running buffer allowed a very fast separation with good resolution. However, only two peaks of stereoisomers were separated, which are cis2R4S and cis-2S4R. The linearity of the method was assessed from 10–100 mg/L for racemic mixture of ketoconazole. The limit of detection (LOD) of each enantiomer was 0.25 mg/L but the limit of quantification (LOQ) was not mentioned. The precision (% RSD) was found to be lower than 1.2, 4.9 and 6.1% for migration, enantiomeric resolution, and corrected peak areas, respectively. The precision of the method was found to be 97.5, 98.5, and 96.0% for the tablets, the syrup, and the gel, respectively. In this current study, we developed an EKC method for the separation of four enantiomers with addition of an anionic surfactant, SDS. Effect of different concentration of TMβCD and buffer solution. In
the first step, the instrumental parameters used were 5 s hydrodynamic injection at 50 mbar pressure, 25 °C of separation temperature, and 25 kV of applied voltage. The concentration of TMβCD was first varied in the range of 3–30 mM using initially 40 mM phosphate buffer solution and acidic pH (pH 3.0) to find the best separation of ketoconazole. We found that only two of the stereoisomers were successfully separated at all concentration ranges used. The migration time increased when the concentration of TMβCD increased and
CHIRAL SEPARATION OF FOUR STEREOISOMERS
improved the resolution of peaks (electropherogram not shown). We chose 20 mM of TMβCD for further study since it gave the highest resolution (Rs =4.24).The influence of phosphate buffer concentrations was investigated in the range 5–50 mM (electropherogram not shown). Changing the buffer concentration only affects the migration time and resolution; still, there were only two enantioseparated peaks observed. Phosphate buffer concentration at 10 mM was preferred for further optimization, as it gave good resolution (Rs =0.98 5.04) with reasonable analysis time (11 13 min). Effect of SDS concentration. The anionic surfactant, SDS, was
added to the buffer solution to enhance the separation of ketoconazole stereoisomers. Theoretically, the hydrophobic tail of SDS monomers will be included in the cyclodextrin (CD) cavity along with the solute. This phenomenon could change the nature of the solute and CD interaction and subsequently change the resolution.15 A previous study reported that the addition of an anionic surfactant, SDS, improved enantioseparation of fenticonazole by increasing the contact time between the chiral compound and chiral selector.16 We optimized the addition of SDS from 3–20 mM to find the best separation of ketoconazole diastereomers. In general, SDS is the selected anionic surfactant and it is used at concentrations basically higher than the CMC.17 The CMC value for SDS in water without any additive has been reported as 8.0 × 10 3 mol L 1. However, when some additive was added, the value of CMC would be decreased.18 In this study, we observed that two smaller peaks appeared at 5 mM SDS concentration. Increasing the SDS concentration increased the migration time of the stereoisomers and reduced the selectivity. The separation with SDS was carried out at low pH (pH 3.0), where the EOF is suppressed. Effective separation of ketoconazole enantiomers in the presence of SDS might be a consequence of the effective ion-pair interactions between ketoconazole enantiomers and SDS monomers where micellar solubilization acts over CMC or probably due to direct electrostatic binding to the negatively charged anionic surfactant, SDS.19 The SDS concentration of 5 mM was selected for further optimization ( electropherogram not shown).
Fig. 2. Electropherograms of enantioseparation of ketoconazole drugs at different percentages of methanol. Separation conditions: 5 mM SDS, 20 mM TMβCD in 10 mM phosphate buffer (pH 2.5); capillary, 64.5 cm × 50 μm I.D. (effective length, 56 cm); voltage, 25 kV; temperature, 25 °C; detection wavelength, 200 nm; hydrodynamic injection, 50 mbar for 5 s; analyte concentra1 tion, 100 mg L ).
Effect of methanol. The organic modifiers frequently affect
the enantiomeric resolution by changing the viscosity of the BGE and the stability of the inclusion complex.8,24 In order to enhance the resolution of enantiomers, methanol as an organic modifier was added to the running buffer solution. Addition of methanol into the BGE successfully separated all four ketoconazole enantiomers, probably due to alteration of the electrophoretic mobilities of the analytes.25 Methanol was added in the range of 0.5 2.0% (v/v). The resolution improved when the percentage of methanol was increased from 0.5% to 1.0% (v/v). However, poor resolution was obtained when 2.0% (v/v) of methanol was added. Therefore, 1.0% (v/v) of methanol was selected as the optimum percentage of organic modifier in running buffer (Fig. 2). Further optimization was carried out by optimizing the separation at different voltages from 20 kV to 28 kV, which basically affects the migration time and the Joule heating. Generally, increasing the voltage gives rise to shorter migration times but is counteracted by the increased Joule heating.24,26 An efficient resolution with good peak shape and reasonable migration time was achieved when the separation was performed at 25 kV (Fig. 3). Increasing the separation voltage to 28 kV decreased the migration time but the resolution become poor, as the peaks overlapped. The temperature of the capillary has an effect on the viscosity of the BGE and kinetic complex formation, which also had a significant effect on the analysis time.24,26 The influence of the temperature (20 30 °C) on the separation of ketoconazole was also studied and the best enantioseparation was obtained at 25 °C (electropherogram not shown).
Effect of different voltage and temperature. Effect of buffer pH. The pH of the buffer solution affects both
the charges on the analytes and the magnitude of the electroosmotic flow (EOF), hence improving the selectivity in CE. The pH of the buffer solution has a significant role in enantioseparation of the drugs, as it may affect the charge of the analytes, and thus the binding characteristics.15,20 The effect of running buffer pH on resolution was studied in the pH range 2.5–9.0 (electropherograms not shown) since the ketoconazole drug has two different pKa values, 6.51 and 2.94. Increasing the pH decreased the migration time as well as the resolution. Based on the results, pH 3.0 provided full resolution of all four diastereomers. However, the efficiency of the peaks was poor since the peak areas are much smaller. In addition, we observed the two smaller peaks of trans-2R4R and trans-2S4S that is not stable at pH 3.0. Therefore, pH 2.5 was selected because the peaks observed have high peak areas as compared to pH 3.0. A previous study proved that EKC21,22 and MEKC23 mode was successfully developed for imidazole and triazole derivatives using an acidic phosphate buffer using uncharged CDs. The uncharged CDs migrate at the same velocity as the EOF, and as a result they only allow the separation of ionized analytes in a pH buffer below the pKa of azole derivatives.21
Precision Studies
In this work, the optimum conditions for separation of the four ketoconazole enantiomers was obtained by using BGE consisting of 20 mM TM βCD, 5 mM SDS, 10 mM phosphate buffer (pH 2.5), 1.0% v/v MeOH at 25 kV separation voltage at 25 °C (Fig. 4). A big difference in peak area between Chirality DOI 10.1002/chir
WAN IBRAHIM ET AL.
precision.27 In this study, poor reproducibility was observed for peak areas, probably due to less stability of the complex formation of ketoconazole drugs with TMβCD. Ketoconazole is a weak base analyte which is positively charged in acidic solutions and electrostatically attracted to the negatively charged capillary walls.28 Consequently, the lower precision is probably due to the irreproducible adsorption, which caused variations in migration time and peak area. Additionally, impurity peaks may be disguised by the peak tailing or resolution of enantiomers, in which two analytes migrating closely after each other may result in poor precision.29 CONCLUSIONS
Fig. 3. Enantioresolution of ketoconazole at different voltages. Separation conditions: 5 mM SDS, 20 mM TMβCD in 10 mM phosphate buffer (pH 2.5), 1.0% methanol (v/v); capillary, 64.5 cm × 50 μm I.D. (effective length, 56 cm); temperature, 25 °C; detection wavelength, 200 nm; hydrodynamic injection, 1 50 mbar for 5 s; analyte concentration, 100 mg L ).
stereoisomers P3, P4 with stereoisomer P1, P2 as observed in the electropherogram is probably due to differences in the percentage of racemic mixture of the compound. Upon optimization of the above-referenced separation conditions, analytical performance of the methods was also investigated for chiral separation of ketoconazole. In this study, the parameter investigated was precision (repeatability and reproducibility). Precision was assessed as relative standard deviation (RSD) for enantioseparation of ketoconazole (100 μg mL-1). Repeatability of three consecutive injections obtained for the developed methods in terms of RSD (n =3) was from 5.42 to 5.80% for migration times, and 10.35 to 14.88% for peak areas. Reproducibility was assessed on 3 different days and on each day triplicate injections were performed. The RSD (n =9) calculated was from 7.15 to 8.88% for migration times, and 7.6 to 25.68% for peak areas. Even though CE offers a broad range of selectivity in combination with high separation efficiency and fast analysis time, CE still has some major problems, such as poor
Fig. 4. Enantioresolution of ketoconazole using optimum CD-EKC conditions. Separation conditions: 5 mM SDS, 20 mM TMβCD in 10 mM phosphate buffer (pH 2.5), 1.0% methanol (v/v); capillary, 64.5 cm × 50 μm I.D. (effective length, 56 cm); voltage, 25 kV; temperature, 25 °C; detection wavelength, 200 nm; hydrodynamic injection, 50 mbar for 5 s; analyte concentration, 1 100 mg L ). Chirality DOI 10.1002/chir
The enantioseparation of four stereoisomers of ketoconazole was successfully performed using CD-EKC. In the present work, the optimum separation of ketoconazole peaks was accomplished with 20 mM TMβCD, 5 mM SDS in 10 mM phosphate buffer solution (pH 2.5), and addition of 1.0% (v/v) of methanol. A separation time within 17 min was achieved under the optimum conditions with acceptable Rs (>1.5). It was shown that the presence of SDS below its CMC value in the BGE greatly influenced resolution and selectivity of the peaks relevant to the enantiomers of ketoconazole when an EKC method was applied using TMβCD in acidic buffer as a chiral selector. Even though there are EKC methods that offer shorter analysis, only two enantiomers of ketoconazole were observed, and thus is not the method for an enantiomeric purity check. The precision of the developed method was measured in terms of repeatability and reproducibility. Poor reproducibility was obtained for the enantioseparation of ketoconazole, probably due to the instability of the drug complexes being formed. ACKNOWLEDGMENTS
The authors thank the Ministry of Education Malaysia (MOE) and Universiti Teknologi Malaysia for the Tier 1 Research University Grant (vote no. 04H22). S.R. Arsad also thanks MOE Malaysia for the MyPhD scholarship. LITERATURE CITED 1. Ali I, Aboul-Enein HY, Gaitonde VD, Singh P, Rawat MSM, Sharma B. Chiral separations of imidazole antifungal drugs on Amy Coat RP column in HPLC. Chromatographia 2009;70(1-2):223–227. 2. Bernal JL, Toribio L, del Nozal MJ, Nieto EM, Montequi MI. Separation of antifungal chiral drugs by SFC and HPLC: a comparative study. J Biochem Biophys Methods 2002;54(1-3):245–254. 3. Terabe S, Otsuka K. Review. Separation techniques of enantiomers by capillary electrophoretic. J Chromatogr A 1994;666:295–319. 4. Nishi H, Terabe S. Optical resolution of drugs by capillary electrophoretic techniques. J Chromatogr A 1995;694(1):245–276. 5. Nishi H. Enantiomer separation of drugs by electrokinetic chromatography. J Chromatogr A 1996;735(1-2):57–76. 6. Rotstein DM, Kertesz DJ, Walker KAM, Swinney DC. Stereoisomers of ketoconazole: preparation and biological activity. J Med Chem 1992;35(15):2818–2825. 7. Saag MS, Dismukes WE. Minireview azole antifungal agents: emphasis. Antimicrob Agents Chemother 1988;32(1):1–8 8. Bernal JL, del Nozal MJ, Toribio L, Montequi MI, Nieto EM. Separation of ketoconazole enantiomers by chiral subcritical fluid chromatography. J Biochem Biophys Methods 2000;43(1-3):241–250. 9. 9.Gübitz G, Schmid MG. Chiral separation by capillary electromigration techniques. J Chromatogr A 2008;1204(2):140–156. 10. 10.Castro-Puyana M, Crego AL, Marina ML. Enantiomeric separation of ketoconazole and terconazole antifungals by electrokinetic
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Chirality DOI 10.1002/chir
Chiral Separation of Four Stereoisomers of Ketoconazole Drugs Using Capillary Electrophoresis WAN AINI WAN IBRAHIM,1,2,3* SITI ROSILAH ARSAD,1,2,3 HASMERYA MAAROF,1,2,3 MOHD. MARSIN SANAGI,1,2,3 4 AND HASSAN Y. ABOUL-ENEIN ** 1 Separation Science & Technology Group (SepSTec), Department of Chemistry, Faculty of Science, Sukdai, Johor Bahru, Johor, Malaysia 2 Nanotechnology Research Alliance, Universiti Teknologi Malaysia, Sukdai, Johor Bahru, Johor, Malaysia 3 Ibnu Sina Institute for Fundamental Science Studies, Universiti Teknologi Malaysia, Sukdai, Johor Bahru, Johor, Malaysia 4 Department of Pharmaceutical and Medicinal Chemistry, Pharmaceutical and Drug Industries Research Division, National Research Centre, Dokki, Cairo, Egypt
ABSTRACT This work aimed to develop a chiral separation method of ketoconazole enantiomers using electrokinetic chromatography. The separation was achieved using heptakis (2, 3, 6tri-O-methyl)-β-cyclodextrin (TMβCD), a commonly used chiral selector (CS), as it is relatively inexpensive and has a low UV absorbance in addition to an anionic surfactant, sodium dodecyl sulfate (SDS). The influence of TMβCD concentration, phosphate buffer concentration, SDS concentration, buffer pH, and applied voltage were investigated. The optimum conditions for chiral separation of ketoconazole was achieved using 10 mM phosphate buffer at pH 2.5 containing 20 mM TMβCD, 5 mM SDS, and 1.0% (v/v) methanol with an applied voltage of 25 kV at 25 °C with a 5-s injection time (hydrodynamic injection). The four ketoconazole stereoisomers were successfully resolved for the first time within 17 min (total analysis time was 28 min including capillary conditioning). The migration time precision of this method was examined to give repeatability and reproducibility with RSDs ≤5.80% (n =3) and RSDs ≤8.88% (n =9), respectively. Chirality 00:000-000, 2014. © 2014 Wiley Periodicals, Inc. KEY WORDS: electrokinetic chromatography; chiral separation; azole drugs; ketoconazole; heptakis (2,3,6-tri-O-methyl)-β-cyclodextrin; sodium dodecyl sulfate INTRODUCTION
Chirality has been and still remains one of the major issues in drug development and the pharmaceutical industry. The United States Food and Drug Administration, European Committee for Proprietary Medicinal Products, and other drug controlling agencies have issued certain guidelines to pharmaceutical and agrochemical industries about the marketing of racemates.1 In the pharmaceutical industry, the separation of enantiomers is growing in interest because the enantiomers of a compound can display quite different activity and toxicity profiles. For this reason, the U.S. Food and Drug Administration has endorsed the development of enantiomerselective synthesis and analysis methods when a new drug is going to be developed and marketed.2 There is no doubt that enantiomer separation by capillary electrophoresis (CE) has rapidly attracted attention as a promising technique due to its high separation efficiency and flexibility. Among various different CE modes, capillary zone electrophoresis (CZE) and electrokinetic chromatography (EKC), in which only a chiral selector is added to the usual running buffer solution, are the most widely used for enantiomer separations.3,4 One of the most attractive advantages of EKC for the separation of enantiomers is an easy change of separation media in the method development; that is, one can easily alter the separation solution to find the optimum separation media and one can also use an expensive chiral selector because of the small amounts required.5 Ketoconazole was the first orally active azole introduced in clinical practice as an effective antifungal agent. Ketoconazole is chiral and has two stereogenic centers in the molecule. Their absolute configuration has been determined via synthesis by Rotstein et al.6 as cis-2R,4S and 2S,4R, also trans-2R4R © 2014 Wiley Periodicals, Inc.
and 2S4S (Fig. 1). The trans diastereomers were found to be a much weaker inhibitor than the cis pair. Earlier chiral separation of ketoconazole was carried out using supercritical fluid chromatography (SFC) and highperformance liquid chromatography (HPLC) on a chiral stationary phase based on substituted polysaccharides with different groups such as 3,5-dimethylphenylcarbamates and (R)-phenyl-ethylcarbamate. The separation of ketoconazole using SFC and HPLC only gives two stereoisomers with Rs =0.72–0.83, k =3.7–12.2 min and Rs =1.53 - 1.70, k =10.8–17.2 min, respectively.7 In the year 2000, the chiral separation of ketoconazole using SFC on an amylosebased column was described. Two stereoisomers of ketoconazole were resolved in less than 7 min with high resolution (Rs =4.29).8 Recently, the chiral separation of ketoconazole was carried out by using EKC, which focused on investigating the applicability of a chiral selector to recognize the enantiomers stereoselectivity. The most widely used chiral selector in EKC are cyclodextrins, due to their low UV absorbance, relative inexpense, ease of use, and ready availablity.9,10 Only two stereoisomers of ketoconazole were successfully resolved using EKC with TMβCD as the chiral selector in a 100 mM *Correspondence to: Wan Aini Wan Ibrahim, Separation Science & Technology Group (SepSTec), Department of Chemistry, Faculty of Science, UTM, 81310 UTM Johor Bahru, Johor, Malaysia. E-mail: [email protected] or [email protected]; Hassan Y. Aboul-Enein, Department of Pharmaceutical and Medicinal Chemistry, National Research Centre, Dokki, Cairo 12311, Egypt. E-mail: [email protected] Received for publication 13 July 2014; Accepted 27 October 2014 DOI: 10.1002/chir.22416 Published online in Wiley Online Library (wileyonlinelibrary.com).
WAN IBRAHIM ET AL.
Fig. 1. Stereoisomers of ketoconazole.
phosphate buffer (pH 3.5). Good precision was obtained, where the RSD was found to be lower than 1.2, 4.9, and 6.1% for migration time, enantiomeric resolution, and corrected peak areas, respectively.10 The use of high buffer concentration is not favorable in CE analysis, as it will generate high current and lead to Joule heating.11–13 To the best of our knowledge, only two stereoisomers of ketoconazole were successfully resolved using SFC, HPLC, and CE. Thus, in this study we modified and optimized the composition of background electrolyte (BGE), which is the key parameter in CE to separate all four stereoisomers of ketoconazole. Our main interest was to apply an anionic surfactant solution, sodium dodecyl sulfate (SDS), in the BGE, which has been the most popular technique for many years. Generally, addition of anionic surfactant solution into BGE at a higher concentration than the critical micelle concentration (CMC) is effective for the separation of ionic analytes and also electrically neutral or nonionic analytes. Thus, the addition of anionic surfactant into BGE is suitable for drug analyses involving cationic, anionic and neutral drugs.14 SDS functions as an anionic selector and also carrier for the neutral analyte ketoconazole. MATERIALS AND METHODS Chemicals and Reagents Ketoconazole was purchased from Dr Ehrenstorfer (Augsburg, Germany) (batch number C14532000, 99.0% purity), heptakis(2,3,6-tri-Omethyl)-β-cyclodextrin (TMβCD) was purchased from Sigma-Aldrich 1 (St. Louis, MO), (batch number H4645-5G, MW: 1429.54 g mol , purity ≥90%), and sodium dodecyl sulfate (SDS) was obtained from Fisher Scientific (Loughborough, UK). Sodium hydroxide (NaOH) and disodium hydrogen phosphate 12-hydrate were purchased from Riedel-de Haen (Seelze, Germany). All other chemicals and solvents were common brands of analytical-reagent grade. Water used for dilutions or as buffer preparation was produced from a Water Purification System from 1 Millipore (Molsheim, France). The stock solution (1000 mg L ) of the ketoconazole was prepared by dissolving the drug in HPLC-grade metha1 nol and diluted to 100 mg L as a typical concentration of sample to be injected. All the separation solutions were filtered through a 0.20-μm nylon syringe filter from Whatman (Clifton, NJ).
Method All electropherograms were obtained with the G1600A Agilent capillary electrophoresis system from Agilent Technologies (Waldbronn, Germany), equipped with temperature control and diode array detection (DAD). Separations were performed using an untreated fused silica capillary of 64.5 cm × 50 μm i.d. (with an effective length of 56 cm to the Chirality DOI 10.1002/chir
detector window) obtained from Polymicro Technologies (Phoenix, AZ). Background electrolytes (BGE) were prepared by dissolving anionic surfactant, SDS, and TMβCD at an adequate concentration in a solution of disodium hydrogen phosphate 12-hydrate where phosphoric acid was used to adjust the preferred pH of the phosphate buffer. Before use, the new capillary was rinsed with 1.0 M NaOH solution for 40 min, 0.1 M NaOH solution for 40 min, with deionized water for 40 min, and finally with the running buffer for 40 min. In between runs, the capillary was conditioned with 0.1 M NaOH for 3 min, followed by deionized water for 3 min and with BGE for 5 min.
RESULTS AND DISCUSSION Method Development
The method development for chiral separation was initially performed by optimizing the experimental parameters such as concentration of chiral selector, concentration of running electrolyte, pH of buffer solution, as well as the use of additives and the presence of an organic modifier. In addition, the effect of different instrumental conditions such as applied voltage and separation temperature was also investigated. In a previous article10 regarding the chiral separation of ketoconazole by EKC, we noted that the use of TMβCD as a chiral selector with a high concentration of running buffer allowed a very fast separation with good resolution. However, only two peaks of stereoisomers were separated, which are cis2R4S and cis-2S4R. The linearity of the method was assessed from 10–100 mg/L for racemic mixture of ketoconazole. The limit of detection (LOD) of each enantiomer was 0.25 mg/L but the limit of quantification (LOQ) was not mentioned. The precision (% RSD) was found to be lower than 1.2, 4.9 and 6.1% for migration, enantiomeric resolution, and corrected peak areas, respectively. The precision of the method was found to be 97.5, 98.5, and 96.0% for the tablets, the syrup, and the gel, respectively. In this current study, we developed an EKC method for the separation of four enantiomers with addition of an anionic surfactant, SDS. Effect of different concentration of TMβCD and buffer solution. In
the first step, the instrumental parameters used were 5 s hydrodynamic injection at 50 mbar pressure, 25 °C of separation temperature, and 25 kV of applied voltage. The concentration of TMβCD was first varied in the range of 3–30 mM using initially 40 mM phosphate buffer solution and acidic pH (pH 3.0) to find the best separation of ketoconazole. We found that only two of the stereoisomers were successfully separated at all concentration ranges used. The migration time increased when the concentration of TMβCD increased and
CHIRAL SEPARATION OF FOUR STEREOISOMERS
improved the resolution of peaks (electropherogram not shown). We chose 20 mM of TMβCD for further study since it gave the highest resolution (Rs =4.24).The influence of phosphate buffer concentrations was investigated in the range 5–50 mM (electropherogram not shown). Changing the buffer concentration only affects the migration time and resolution; still, there were only two enantioseparated peaks observed. Phosphate buffer concentration at 10 mM was preferred for further optimization, as it gave good resolution (Rs =0.98 5.04) with reasonable analysis time (11 13 min). Effect of SDS concentration. The anionic surfactant, SDS, was
added to the buffer solution to enhance the separation of ketoconazole stereoisomers. Theoretically, the hydrophobic tail of SDS monomers will be included in the cyclodextrin (CD) cavity along with the solute. This phenomenon could change the nature of the solute and CD interaction and subsequently change the resolution.15 A previous study reported that the addition of an anionic surfactant, SDS, improved enantioseparation of fenticonazole by increasing the contact time between the chiral compound and chiral selector.16 We optimized the addition of SDS from 3–20 mM to find the best separation of ketoconazole diastereomers. In general, SDS is the selected anionic surfactant and it is used at concentrations basically higher than the CMC.17 The CMC value for SDS in water without any additive has been reported as 8.0 × 10 3 mol L 1. However, when some additive was added, the value of CMC would be decreased.18 In this study, we observed that two smaller peaks appeared at 5 mM SDS concentration. Increasing the SDS concentration increased the migration time of the stereoisomers and reduced the selectivity. The separation with SDS was carried out at low pH (pH 3.0), where the EOF is suppressed. Effective separation of ketoconazole enantiomers in the presence of SDS might be a consequence of the effective ion-pair interactions between ketoconazole enantiomers and SDS monomers where micellar solubilization acts over CMC or probably due to direct electrostatic binding to the negatively charged anionic surfactant, SDS.19 The SDS concentration of 5 mM was selected for further optimization ( electropherogram not shown).
Fig. 2. Electropherograms of enantioseparation of ketoconazole drugs at different percentages of methanol. Separation conditions: 5 mM SDS, 20 mM TMβCD in 10 mM phosphate buffer (pH 2.5); capillary, 64.5 cm × 50 μm I.D. (effective length, 56 cm); voltage, 25 kV; temperature, 25 °C; detection wavelength, 200 nm; hydrodynamic injection, 50 mbar for 5 s; analyte concentra1 tion, 100 mg L ).
Effect of methanol. The organic modifiers frequently affect
the enantiomeric resolution by changing the viscosity of the BGE and the stability of the inclusion complex.8,24 In order to enhance the resolution of enantiomers, methanol as an organic modifier was added to the running buffer solution. Addition of methanol into the BGE successfully separated all four ketoconazole enantiomers, probably due to alteration of the electrophoretic mobilities of the analytes.25 Methanol was added in the range of 0.5 2.0% (v/v). The resolution improved when the percentage of methanol was increased from 0.5% to 1.0% (v/v). However, poor resolution was obtained when 2.0% (v/v) of methanol was added. Therefore, 1.0% (v/v) of methanol was selected as the optimum percentage of organic modifier in running buffer (Fig. 2). Further optimization was carried out by optimizing the separation at different voltages from 20 kV to 28 kV, which basically affects the migration time and the Joule heating. Generally, increasing the voltage gives rise to shorter migration times but is counteracted by the increased Joule heating.24,26 An efficient resolution with good peak shape and reasonable migration time was achieved when the separation was performed at 25 kV (Fig. 3). Increasing the separation voltage to 28 kV decreased the migration time but the resolution become poor, as the peaks overlapped. The temperature of the capillary has an effect on the viscosity of the BGE and kinetic complex formation, which also had a significant effect on the analysis time.24,26 The influence of the temperature (20 30 °C) on the separation of ketoconazole was also studied and the best enantioseparation was obtained at 25 °C (electropherogram not shown).
Effect of different voltage and temperature. Effect of buffer pH. The pH of the buffer solution affects both
the charges on the analytes and the magnitude of the electroosmotic flow (EOF), hence improving the selectivity in CE. The pH of the buffer solution has a significant role in enantioseparation of the drugs, as it may affect the charge of the analytes, and thus the binding characteristics.15,20 The effect of running buffer pH on resolution was studied in the pH range 2.5–9.0 (electropherograms not shown) since the ketoconazole drug has two different pKa values, 6.51 and 2.94. Increasing the pH decreased the migration time as well as the resolution. Based on the results, pH 3.0 provided full resolution of all four diastereomers. However, the efficiency of the peaks was poor since the peak areas are much smaller. In addition, we observed the two smaller peaks of trans-2R4R and trans-2S4S that is not stable at pH 3.0. Therefore, pH 2.5 was selected because the peaks observed have high peak areas as compared to pH 3.0. A previous study proved that EKC21,22 and MEKC23 mode was successfully developed for imidazole and triazole derivatives using an acidic phosphate buffer using uncharged CDs. The uncharged CDs migrate at the same velocity as the EOF, and as a result they only allow the separation of ionized analytes in a pH buffer below the pKa of azole derivatives.21
Precision Studies
In this work, the optimum conditions for separation of the four ketoconazole enantiomers was obtained by using BGE consisting of 20 mM TM βCD, 5 mM SDS, 10 mM phosphate buffer (pH 2.5), 1.0% v/v MeOH at 25 kV separation voltage at 25 °C (Fig. 4). A big difference in peak area between Chirality DOI 10.1002/chir
WAN IBRAHIM ET AL.
precision.27 In this study, poor reproducibility was observed for peak areas, probably due to less stability of the complex formation of ketoconazole drugs with TMβCD. Ketoconazole is a weak base analyte which is positively charged in acidic solutions and electrostatically attracted to the negatively charged capillary walls.28 Consequently, the lower precision is probably due to the irreproducible adsorption, which caused variations in migration time and peak area. Additionally, impurity peaks may be disguised by the peak tailing or resolution of enantiomers, in which two analytes migrating closely after each other may result in poor precision.29 CONCLUSIONS
Fig. 3. Enantioresolution of ketoconazole at different voltages. Separation conditions: 5 mM SDS, 20 mM TMβCD in 10 mM phosphate buffer (pH 2.5), 1.0% methanol (v/v); capillary, 64.5 cm × 50 μm I.D. (effective length, 56 cm); temperature, 25 °C; detection wavelength, 200 nm; hydrodynamic injection, 1 50 mbar for 5 s; analyte concentration, 100 mg L ).
stereoisomers P3, P4 with stereoisomer P1, P2 as observed in the electropherogram is probably due to differences in the percentage of racemic mixture of the compound. Upon optimization of the above-referenced separation conditions, analytical performance of the methods was also investigated for chiral separation of ketoconazole. In this study, the parameter investigated was precision (repeatability and reproducibility). Precision was assessed as relative standard deviation (RSD) for enantioseparation of ketoconazole (100 μg mL-1). Repeatability of three consecutive injections obtained for the developed methods in terms of RSD (n =3) was from 5.42 to 5.80% for migration times, and 10.35 to 14.88% for peak areas. Reproducibility was assessed on 3 different days and on each day triplicate injections were performed. The RSD (n =9) calculated was from 7.15 to 8.88% for migration times, and 7.6 to 25.68% for peak areas. Even though CE offers a broad range of selectivity in combination with high separation efficiency and fast analysis time, CE still has some major problems, such as poor
Fig. 4. Enantioresolution of ketoconazole using optimum CD-EKC conditions. Separation conditions: 5 mM SDS, 20 mM TMβCD in 10 mM phosphate buffer (pH 2.5), 1.0% methanol (v/v); capillary, 64.5 cm × 50 μm I.D. (effective length, 56 cm); voltage, 25 kV; temperature, 25 °C; detection wavelength, 200 nm; hydrodynamic injection, 50 mbar for 5 s; analyte concentration, 1 100 mg L ). Chirality DOI 10.1002/chir
The enantioseparation of four stereoisomers of ketoconazole was successfully performed using CD-EKC. In the present work, the optimum separation of ketoconazole peaks was accomplished with 20 mM TMβCD, 5 mM SDS in 10 mM phosphate buffer solution (pH 2.5), and addition of 1.0% (v/v) of methanol. A separation time within 17 min was achieved under the optimum conditions with acceptable Rs (>1.5). It was shown that the presence of SDS below its CMC value in the BGE greatly influenced resolution and selectivity of the peaks relevant to the enantiomers of ketoconazole when an EKC method was applied using TMβCD in acidic buffer as a chiral selector. Even though there are EKC methods that offer shorter analysis, only two enantiomers of ketoconazole were observed, and thus is not the method for an enantiomeric purity check. The precision of the developed method was measured in terms of repeatability and reproducibility. Poor reproducibility was obtained for the enantioseparation of ketoconazole, probably due to the instability of the drug complexes being formed. ACKNOWLEDGMENTS
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Chirality DOI 10.1002/chir
