CHIRALITY 26:394–399 (2014)
Application of Maltodextrin as Chiral Selector in Capillary Electrophoresis for Quantification of Amlodipine Enantiomers in Commercial Tablets SAEED NOJAVAN,1* SHABNAM POURMOSLEMI,2 HAMIDEH BEHDAD,1 ALI REZA FAKHARI,1 AND ALI MOHAMMADI2,3* 1 Faculty of Chemistry, Shahid Beheshti University, Evin, Tehran, Iran 2 Department of Drug & Food Control, Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran, Iran 3 Pharmaceutical Quality Assurance Research Center, Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran, Iran
ABSTRACT Maltodextrin was investigated as a chiral selector in capillary electrophoresis (CE) analysis of amlodipine (AM) enantiomers. For development of a stereoselective CE method, various effective parameters on the enantioseparation were optimized. The best results were achieved on an uncoated fused silica capillary at 20 °C using phosphate buffer (100 mM, pH 4) containing 10% w/v maltodextrin (dextrose equivalent value 4–7). The UV detector was set at 214 nm and a constant voltage of 20 kV was applied. The range of quantitation was 2.5–250 μg/mL (R2 > 0.999) for both enantiomers. Intra- (n = 5) and interday (n = 3) relative standard deviation (RSD) values were less than 7%. The limits of quantitation and detection were 1.7 μg/mL and 0.52 μg/mL, respectively. Recoveries of R(+) and S( ) enantiomers from tablet matrix were 97.2% and 97.8%, respectively. The method was applied for the quantification of AM enantiomers in commercial tablets. Also, the enantioseparation capability of heparin was evaluated and the results showed that heparin did not have any chiral selector activity in this study. Chirality 26:394–399, 2014. © 2014 Wiley Periodicals, Inc. KEY WORDS: amlodipine; capillary electrophoresis; enantioseparation; maltodextrin; heparin INTRODUCTION
Amlodipine (AM), 2-[(2-aminoethoxy)-methyl]-4-(2-chlorophenyl)1,4-dihydro-6-methyl-3,5-pyridine-dicarboxylic acid 3-ethyl 5-methyl ester (Fig. 1), is a dihydropyridine calcium-channel blocker.1,2 AM inhibits the transmembrane influx of calcium ions into vascular smooth muscle and cardiac muscle cells and is used in the management of hypertension and angina pectoris.3 Like most other calcium blocker agents of the dihydropyridine type, AM is therapeutically used as the racemic mixture of R(+) and S( ) enantiomers. However, the S( ) enantiomer has shown to be more than 2000 times more potent than the R(+) enantiomer in calcium channel blocking activity.4,5 As a result of different pharmacologic and toxicologic properties of enantiomers, enantioselective analytical and preparative methods are required not only for pharmacodynamic and pharmacokinetic studies, but also for quality control of pharmaceutical preparations and determination of their enantiomeric purity.6 Chromatography and electromigration techniques have long been the methods of choice for enantioseparation analysis.7 In the last years, capillary electrophoresis (CE) has been increasingly applied for the enantioseparation of pharmaceutical compounds, due to its several attractive features such as speed of method development and analysis, robustness, simplicity, and low cost. Typical enantiomer separations by CE are performed by simply applying chiral additives to the running buffer of CE. Polysaccharides, proteins, crown ethers, and chiral surfactants are some of the chiral additives that have been used for CE enantiomer separation studies.8 Recently, liquid chromatography-mass spectrometry (LC-MS) was applied for quantification of AM enantiomers in human plasma.9 Also, AM enantiomers were separated on a molecularly imprinted polymer-based stationary phase.10 In another work, chiral LC was used for determination of enantiomeric purity of S( )-AM.11 AM enantiomers have been separated by a number © 2014 Wiley Periodicals, Inc.
of CE methods, most of them using cyclodextrins (CDs) as chiral selectors. Five neutral CDs (α-CD, β-CD, γ-CD, hydroxypropyl-βCD [HP-β-CD] and hydroxyethyl-β-CD) and the anionic sulphobutylether-β-CD and carboxymethyl-β-CD have been examined for the resolution of AM enantiomers in both highperformance liquid chromatography (HPLC) and CE.12 In different reports, hydroxypropyl-α-CD (HP-α-CD) and HP-β-CD modified CE procedures were used for quantification of AM enantiomers in commercial tablets, human plasma, and serum samples.2,13–16 Also, polybrene, a cationic polymer, was used for improvement of AM enantioseparation using CE.17 An on-line coupled isotachophoresis-capillary zone electrophoresis (CZE) separation method using HP-β-CD as chiral selector enabled the direct detection and quantitation of AM enantiomers in urine samples.18 Dual chiral selector systems consisting of glycogen (neutral polysaccharide) with chondroitin sulfate A (ionic polysaccharide), β-CD, and HP-β-CD exhibited good enantioselective properties for AM analysis.19 Recently, polysaccharide-based chiral columns were applied for enantiomer separation of dihydropyridine derivatives.20 In another work, enantiomeric separation of AM and its two chiral impurities by nano-LC and capillary electrochromatography (CEC) using a chiral stationary phase based on cellulose tris(4-chloro-3methylphenylcarbamate) was investigated.21 While CDs are cyclic oligomers, produced from starch by means of enzymatic conversion, maltodextrins and heparin are other polysaccharide-based compounds that have been *Correspondence to: Ali Mohammadi or Saeed Nojavan, Department of Drug & Food Control, Faculty of Pharmacy, Tehran University of Medical Sciences, P.O. Box 14155–6451, Tehran 14174, Iran and Faculty of Chemistry, Shahid Beheshti University, G. C., P.O. Box 19396–4716, Evin, Tehran, Iran. E-mail: [email protected] or [email protected] Received for publication 9 January 2014; Accepted 28 March 2014 DOI: 10.1002/chir.22334 Published online 16 May 2014 in Wiley Online Library (wileyonlinelibrary.com).
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DETERMINATION OF AMLODIPINE ENANTIOMERS
All solutions were prepared by HPLC-grade water that was obtained from a Milli-Q system (Millipore Milford, MA). Buffer and all solutions were filtered through 0.45-μm PTFE membrane filters before use.
CE Equipment Fig. 1. Chemical structure of AM. The asterisk denotes the chiral center.
investigated for their chiral selector properties. Maltodextrins are complex malto-oligo and polysaccharide mixtures obtained from partial acid and/or enzymatic hydrolysis of starch. They are characterized by their dextrose equivalent (DE), which is the equivalent of the degree of polymerization of malto-oligosaccharides. The higher the DE number, the higher is the extent of starch hydrolysis and, consequently, the shorter are the oligomeric chains present in a mixture. Different interactions of chiral solutes with the helical structure of the maltodextrin emerge as the basis of the enantioselectivity. When used in CE, maltodextrins have shown highly efficient chiral selectivity for a broad range of acidic and basic compounds. They were used in the design of potentiometric, enantioselective membrane electrodes for analysis of captopril and baclofen.22,23 Chiral separation of cetirizine and pentazocine was achieved by CE methods using maltodextrins.24,25 Maltodextrin was also used for stability evaluation of tramadol enantiomers using a chiral stability indicating CE method.26 Heparin is a glycosaminoglycan, a heterogeneous mixture of variably sulfated polysaccharide chains. The large but variable degree of sulfation results in heparin being one of the most anionic biopolymers. The highly anionic character of heparin enhances its solubility, while offering the potential for considerable electrophoretic mobility. The electrophoretic mobility of heparin, coupled with its helicity in conjunction with the chirality inherent in the constituent monosaccharide residues suggested it as a chiral mobile phase additive in CE analysis and was successfully used for CE enantioseparation of several antimalarial drugs, antihistamines, and other pharmaceutical compounds.27,28 In the present study, we evaluated the applicability of maltodextrins with different DE values and heparin in order to develop a chiral CE analysis method for AM in commercial tablets. Other contributing factors such as the chiral selector concentration, pH of background electrolyte (BGE), BGE concentration, voltage, and cartridge temperature were optimized and the method was validated and proved to be precise, accurate, and enantioselective enough to be used for quality control of AM enantiomers in tablet dosage form. Using maltodextrin as a commercial chiral selector made this method an inexpensive alternative for previous CE methods, which are usually based on CDs or noncommercial chiral selectors. EXPERIMENTAL Reagents and Materials AM besylate racemic powder and its R(+) and S( ) enantiomers (purity >99.90%) were obtained from Tofigh Daru Research & Engineering Company (Tehran, Iran). AM besylate tablets (Amlotidi 5 mg) made by Tolidaru Pharmaceutical Company (Tehran, Iran) were used for assay studies. Maltodextrins with three different DE values (4–7, 13–17, and 16.5–19.5) were purchased from Sigma-Aldrich Chemie (Steinheim, Germany). Hydrochloric acid, sodium hydroxide, phosphoric acid, Na2HPO4, and NaH2PO4.2H2O salts were purchased from Merck (Darmstadt, Germany). All chemicals and reagents were of analytical grade and were used as received.
CE experiments were carried out using a Lumex Capel 105 (Lumex, St. Petersburg, Russia) equipped with a UV detector, set at 214 nm and an uncoated fused-silica capillary (Lumex), 57 cm × 75 μm I.D. (49 cm effective length). The cartridge temperature was maintained at the desired amount by a water-circulating system. Capillary conditioning was performed daily before the first experiment as follows: 20 min with 0.5 M HCl, 5 min with water, 30 min with 0.5 M NaOH, and 5 min with water. Additionally, the capillary was washed for 2 min with water, 2 min with 0.5 M NaOH, 2 min with water, and 2 min with the running buffer with positive pressure applied at the injection end before each run. Acquisition of electropherograms was computer-controlled by Chrom&Spec software v. 1.5. The analytes were injected at the anodic end by applying the pressure of 60 mbar for 6 s.
Preparation of Stock and Standard Solutions A stock solution of racemic AM (1000 μg/mL) was prepared in water. Stock solutions were protected from light and stored for 3 weeks at 4°C without any decomposition. Aliquots of AM standard stock solution were transferred into 100-mL volumetric flasks and solutions were made up to volume with water to yield final concentrations of 1, 2.5, 5, 10, 25, 50, 100, 150, 200, and 250 μg/mL for each enantiomer.
Preparation of Tablets for Assay Twenty AM tablets were weighed, crushed, and mixed in a mortar and pastel for 20 min. A portion of powder equivalent to the weight of one tablet was accurately weighed and transferred into a 25-mL A-grade volumetric flask and 5 mL water was added to the flask. The volumetric flask was sonicated for 20 min until complete dissolution of the drug. The solution was then made up to volume with HPLC-grade water. An aliquot of the solution was filtered through a 0.45-μm nylon filter and was transferred to a 10-mL A-grade volumetric flask and made up to volume with water to yield a concentration of each enantiomer in the range of linearity.
RESULTS AND DISCUSSION Method Development and Optimization Using Maltodextrin Effect of BGE pH on the enantioseparation. The BGE pH is one
of the most important parameters in CE analysis because of its effect on the electroosmotic flow (EOF) and ionization state of the analyte.29 The AM chemical structure consists of a free amino group with pKa 8.6, which means the molecule is positively charged at pH values under 8.6 and neutral at higher pH values.30 Therefore, it can be deduced that AM is electrophoretically separable at neutral and acidic pH values by normal phase CE methods. EOF is at its lowest amount at pH values below 2 and increases gradually with an increase in pH, which results in shorter migration times and decreased resolution. Different pH values in the range of 2–5 were investigated in order to obtain the optimum amount based on the enantioresolution and rational migration time (Table 1). Although the two enantiomers were best resolved at pH 2, pH 4 was chosen as the optimum pH for further experiments because shorter migration times were obtained for enantiomers and the resolution was not affected significantly. The migration order of each enantiomer was identified using the pure R(+) or S( )-enantiomer. The results showed that R(+)-enantiomer migrated faster than the S( )-enantiomer. Effect of maltodextrin DE value on the enantioseparation. Malto-
dextrins with different DE values (4–7, 13–17, and 16.5–19.5) were evaluated for their chiral selector activity and, as shown Chirality DOI 10.1002/chir
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TABLE 1. Effect of BGE pH on the migration times and resoa lution of AM enantiomers
sites to interact with the analyte molecules which can explain the observed results.24
pH
Effect of maltodextrin concentration on the enantioseparation. Malto-
2 3 4 5
t(R+) (min)
t(S-) (min)
Resolution
34.18 29.88 24.78 20.82
35.01 30.68 25.17 21.18
1.61 1.59 1.45 1.39
a
Experimental condition: capillary column: 57 cm (49 cm effective length), 75 μm id; UV detection: 214 nm; applied voltage: 20 kV; temperature: 25 °C; injection: 6 s (at a pressure of 60 mbar); concentration of each enantiomer: 25 μg/mL, BGE: 100 mM phosphate buffer, containing 10% w/v maltodextrin (DE value 4–7).
in Fig. 2, maltodextrin with DE value 4–7 separated the enantiomers with better resolution and longer migration times. Since maltodextrin is a neutral polysaccharide, hydrophobic and hydrogen bonds are the most important interactions responsible for separating the enantiomers and maltodextrins with lower DE values and longer oligomeric chains have more
dextrin was added to the phosphate buffer at four different concentrations (5, 10, 15, and 20% w/v) and its effects on the migration times and resolution of AM enantiomers were investigated. It was observed that increasing the maltodextrin concentration induced improvement of enantioresolution through the elongation of the migration times. Higher maltodextrin concentrations introduced more interaction sites for AM molecules that resulted in better separation of the enantiomers. Also, increasing of maltodextrin concentration increased the BGE viscosity, which was another reason for the elongated migration times. This effect also prevented us from increasing the maltodextrin concentration above 20% w/v. In order to obtain rational resolution and migration times, 10% w/v was chosen as the optimum maltodextrin concentration (Fig. 3A). Effect of BGE concentration on the enantioseparation. Phosphate
buffer was investigated at four different concentrations; (50, 75, 100, and 120 mM). This parameter affects the migration time by decreasing the EOF and increasing the viscosity and improves the resolution by providing better interactions between AM molecules and maltodextrin through inhibiting their adsorption on the capillary interior wall. According to the data presented in Table 2 and to maintain a balance between resolution and migration time, 100 mM was chosen as the optimum concentration for the phosphate buffer. Effect of applied voltage on the enantioseparation. The effect of
applied voltage on chiral CE analysis can be discussed from different aspects. Increased amounts of voltage usually improve the CE analysis efficiency, decrease the migration times by affecting both EOF and electrophoretic flow, and have a
Fig. 2. Effect of maltodextrin DE value on the migration times and resolution of AM enantiomers: (A) DE value 16.5–19.5; (B) DE value 13–17 and (C) DE value 4–7. Experimental conditions: capillary column: 57 cm (49 cm effective length), 75 μm id; UV detection: 214 nm; applied voltage: 20 kV; temperature: 25 °C; injection: 6 s (at a pressure of 60 mbar); concentration of each enantiomer: 25 μg/mL, BGE: 100 mM phosphate buffer, pH 4.0 containing 10% w/v maltodextrin. Chirality DOI 10.1002/chir
Fig. 3. Effect of (A) maltodextrin concentration and (B) applied voltage on the enantioseparation of AM enantiomers. Experimental conditions: capillary column: 57 cm (49 cm effective length), 75 μm id; UV detection: 214 nm; applied voltage: 20 kV; temperature: 25 °C; injection: 6 s (at a pressure of 60 mbar); concentration of each enantiomer: 25 μg/mL, BGE: 100 mM phosphate buffer, pH 4.0, DE value of maltodextrin: 4–7. In (B) the concentration of maltodextrin was 10% w/v.
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TABLE 2. Effect of buffer concentration on resolution of a amlodipine enantiomers Phosphate buffer concentration (mM)
t(R+) (min)
t(S-) (min)
Resolution
20.23 22.47 24.78 27.63
20.43 22.75 25.17 28.12
0.92 1.1 1.45 1.54
50 75 100 120 a
Experimental condition: capillary column: 57 cm (49 cm effective length), 75 μm id; UV detection: 214 nm; applied voltage: 20 kV; temperature: 25 °C; injection: 6 s (at a pressure of 60 mbar); concentration of each enantiomer: 25 μg/mL, BGE: phosphate buffer, pH 4.0 containing 10% maltodextrin (DE value 4–7).
significant role in Joule heating through increasing the electric current. Voltage was investigated in the range of 12–22 kV in order to reach an optimum amount for separating AM enantiomers. Decreasing the voltage from 22 to 15 kV increased the resolution probably because of decreasing the migration times and providing enough time for interaction of AM and maltodextrin molecules. However, further reduction in voltage from 15 to 12 kV decreased the resolution, which shows the importance of voltage in CE analysis efficiency. Thus, an applied voltage of 20 kV was chosen as the optimum voltage in order to obtain a good resolution with reasonable migration times (Fig. 3B). Effect of cartridge temperature on the enantioseparation. The car-
tridge temperature can affect the EOF and electrophoretic flow through changing the BGE viscosity, which also explains its effect on the resolution. Several temperatures in the range of 15–30 °C were examined in order to find the optimum temperature at which there is a good resolution and reasonable migration times. Increasing the temperature from 15–30 °C, decreased the migration times and resolution due to shorter time for essential interactions. Although the best resolution was achieved at 15 °C, the temperature of 20 °C was chosen as the optimum temperature because of short migration time of each enantiomer in this temperature (Table 3). Method Development and Optimization Using Heparin Evaluation of enantioseparation capability of heparin. Previous
studies have shown that enantioseparation using heparin as a chiral selector can be performed in either normal or reversed polarity.27,28 In this study, at normal polarity, adding heparin into the BGE with pH values in the range of 2–8 did not result in any enantioseparation.
TABLE 3. Effect of cartridge temperature on the migration a times and resolution of amlodipine enantiomers Temperature (°C) 15 20 25 30 a
t(R+) (min)
t(S-) (min)
Resolution
31.17 25.71 22.91 18.18
31.65 26.19 23.26 18.49
1.94 1.68 1.45 1.30
Experimental condition: capillary column: 57 cm (49 cm effective length), 75 μm id; UV detection: 214 nm; voltage: 20 kV; injection: 6 s (at a pressure of 60 mbar); concentration of each enantiomer: 25 μg/mL, BGE: 100 mM phosphate buffer, pH 4.0 containing 10% w/v maltodextrin (DE value 4–7).
Enantioseparation by heparin has also been performed at reverse polarity.28 This experiment was performed by adding heparin into the BGE pH 2. AM was not detected in the resultant electropherogram. This observation together with the normal CE analysis results indicated that there are no interactions between AM and heparin molecules. Stalcup and Agyei,27 who investigated the enantioseparation of several compounds using heparin, concluded that the analyte molecular structure and size are important factors in heparin recognition ability. It seems that the AM molecule does not have the required criteria to produce efficient interactions with heparin. Higher heparin concentrations up to 6% w/v were also investigated to see whether they can make the desired interactions with AM molecules. Increasing the heparin concentration did not have any effect on the enantioseparation but increased the migration times, probably because of the BGE viscosity. Effect of heparin on the enantioseparation in the presence of other chiral selectors. Heparin was added to the BGE containing
maltodextrin or HP-α-CD, and their overall enantioseparation activity was investigated. Maltodextrin and HP-α-CD could resolve AM enantiomers; however, adding 1% w/v heparin deteriorated their chiral selector activity. These results showed that heparin was not only unable to separate AM enantiomers, but also decreased the efficiency of other chiral selector compounds by blocking their interaction sites. Validation of the Method
After choosing maltodextrin as the optimum chiral selector and optimizing various parameters that affect its efficiency, the method was validated for linearity, limit of detection (LOD), limit of quantitation (LOQ), precision, accuracy, and recovery. Linearity. Plots of peak area (Y) against concentration
(X, μg/mL) were drawn for both AM enantiomers. Both plots were linear in the range of 2.5–250 μg/mL and regression equations were calculated as Y = 0.80 X + 3.92 for R(+)-enantiomer and Y = 0.88 X + 3.32 for S( )-enantiomer. Correlation coefficients (R2) were 0.9991 and 0.9992 for R(+) and S( )enantiomers, respectively. LOD and LOQ. According to the pharmacopeial recommen-
dations (United States Pharmacopeia, 2013), the LOD and LOQ were determined for both enantiomers, at signal-tonoise ratios of 3:1 and 10:1, respectively. The LOQ value (1.7 μg/mL) was verified by its nearness to the lower concentration of the working range. The LOD (0.52 μg/mL) was then calculated by dividing LOQ by 3.3. Interday precision was assessed by preparing standard samples with 25 and 250 μg/mL concentrations for each enantiomer and analyzing them in three different days. Intraday precision was assessed by analyzing the above-mentioned standard samples five times in a single day. Relative standard deviations (RSD) are presented in Table 4. We also calculated each enantiomer concentration and relative error (RE) using the average peak areas and the regression equations, which show the method accuracy.
Precision and accuracy.
Recovery. The recovery test was performed in order to inves-
tigate the possible interactions of tablet matrix with analysis of the drug substance. A known amount of standard powder was added to the sample of tablet powder and diluted to yield a concentration of 200 μg/mL for each enantiomer. This sample was analyzed using the optimized method five times. Chirality DOI 10.1002/chir
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TABLE 4. Intra- and interday precision and accuracy of AM enantiomers Found concentration (μg/mL), RSD (%), RE (%) Enantiomer
Actual concentration (μg/mL)
R-(+)
25 250 25 250
S-( )
Intraday 25.47, 249.06, 25.18, 248.85,
Interday
5.1, +1.88 1.8, 0.38 6.1, +0.72 1.9, 0.46
25.55, 5.1, +2.2 247.71, 1.9, 0.92 25.66, 7, +2.64 248.4, 2.1, 0.64
LITERATURE CITED
Fig. 4. Assay of AM R(+) and S( )-enantiomers in a commercial tablet. Experimental conditions: capillary column: 57 cm (49 cm effective length), 75 μm id; UV detection: 214 nm; temperature: 20 °C; voltage: 20 kV; injection: 6 s (at a pressure of 60 mbar); BGE: 100 mM phosphate buffer pH 2 containing 10% w/v maltodextrin (DE value 4–7).
The observed concentrations of R(+) and S( )-enantiomers were found to be 194.45 ± 3.56 μg/mL (mean ± SD) and 195.57 ± 3.81 μg/mL, respectively. The resultant % RSD of these studies were found to be 1.83 % for R(+)-enantiomer and 1.95 % for S( )-enantiomer with the corresponding recovery values of 97.2 % and 97.8 %, respectively. These results indicated that the method was selective for the analysis of both enantiomers without any interference from the excipients used in the formulation and production of the tablets. Assay of AM enantiomers in tablets. The optimized and vali-
dated CE method was used for analysis of AM enantiomers in commercial AM tablets. The amounts of AM R(+) and S ( )-enantiomers were found to be 102.5% (% RSD = 2.1) and 109.3% (% RSD = 5.2) of the label claimed amount through five analyses. The actual amounts are therefore 2.56 mg for R(+)enantiomer and 2.73 mg for S( )-enantiomer. Fig. 4 shows one of the assay electropherograms. CONCLUSIONS
Maltodextrin, a neutral polysaccharide, as a chiral selector was investigated for separation of AM enantiomers. All maltodextrins with different DE values separated AM enantiomers, while the one with DE value 4–7 showed the best results and was used for development and validation of a stereoselective CE method for determination of AM enantiomers in commercial tablets. The method was optimized for its various parameters and produced good resolution between AM enantiomer peaks in a reasonable time. Also, enantioseparation capability of heparin was investigated and the results showed that heparin did not have any chiral selector activity in this study. ACKNOWLEDGMENT
The authors acknowledge the financial assistance from Tehran University of Medical Sciences, Tehran, Iran. The authors thank Shahid Beheshti University for the supply of analytical instruments. Chirality DOI 10.1002/chir
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24. Watanabe T, Takahashi K, Horiuchi M, Kato K, Nakazawa H, Sugimoto T, Kanazawa H. Chiral separation and quantitation of pentazocine enantiomers in pharmaceuticals by capillary zone electrophoresis using maltodextrins. J Pharm Biomed Anal 1999;21:75–81. 25. Nojavan S, Fakhari AR. Chiral separation and quantitation of cetirizine and hydroxyzine by maltodextrin-mediated CE in human plasma: Effect of zwitterionic property of cetirizine on enantioseparation. Electrophoresis 2011a;32:764–771. 26. Mohammadi A, Nojavan S, Rouini M, Fakhari AR. Stability evaluation of tramadol enantiomers using a chiral stability-indicating capillary electrophoresis method and its application to pharmaceutical analysis. J Sep Sci 2011b;34:1613–1620. 27. Stalcup AM, Agyei NM. Heparin: A chiral mobile-phase additive for capillary zone electrophoresis. Anal Chem 1994;66:3054–3059. 28. Jin Y, Stalcup AM. Application of heparin to chiral separations of antihistamines by capillary electrophoresis. Electrophoresis 1998;19:2119–2123. 29. Li C, Jiang Y. Analysis of repaglinide enantiomers in pharmaceutical formulations by capillary electrophoresis using 2,6-Di-o-methyl-β-cyclodextrin as a chiral Selector. J Chromatogr Sci 2012;50:739–743. 30. Moffat AC, Osselton DM, Widdop B. Clarke’s analysis of drugs and poisons: in pharmaceuticals, body fluids, and postmortem material. London: Pharmaceutical Press; 2004.
Chirality DOI 10.1002/chir
Application of Maltodextrin as Chiral Selector in Capillary Electrophoresis for Quantification of Amlodipine Enantiomers in Commercial Tablets SAEED NOJAVAN,1* SHABNAM POURMOSLEMI,2 HAMIDEH BEHDAD,1 ALI REZA FAKHARI,1 AND ALI MOHAMMADI2,3* 1 Faculty of Chemistry, Shahid Beheshti University, Evin, Tehran, Iran 2 Department of Drug & Food Control, Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran, Iran 3 Pharmaceutical Quality Assurance Research Center, Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran, Iran
ABSTRACT Maltodextrin was investigated as a chiral selector in capillary electrophoresis (CE) analysis of amlodipine (AM) enantiomers. For development of a stereoselective CE method, various effective parameters on the enantioseparation were optimized. The best results were achieved on an uncoated fused silica capillary at 20 °C using phosphate buffer (100 mM, pH 4) containing 10% w/v maltodextrin (dextrose equivalent value 4–7). The UV detector was set at 214 nm and a constant voltage of 20 kV was applied. The range of quantitation was 2.5–250 μg/mL (R2 > 0.999) for both enantiomers. Intra- (n = 5) and interday (n = 3) relative standard deviation (RSD) values were less than 7%. The limits of quantitation and detection were 1.7 μg/mL and 0.52 μg/mL, respectively. Recoveries of R(+) and S( ) enantiomers from tablet matrix were 97.2% and 97.8%, respectively. The method was applied for the quantification of AM enantiomers in commercial tablets. Also, the enantioseparation capability of heparin was evaluated and the results showed that heparin did not have any chiral selector activity in this study. Chirality 26:394–399, 2014. © 2014 Wiley Periodicals, Inc. KEY WORDS: amlodipine; capillary electrophoresis; enantioseparation; maltodextrin; heparin INTRODUCTION
Amlodipine (AM), 2-[(2-aminoethoxy)-methyl]-4-(2-chlorophenyl)1,4-dihydro-6-methyl-3,5-pyridine-dicarboxylic acid 3-ethyl 5-methyl ester (Fig. 1), is a dihydropyridine calcium-channel blocker.1,2 AM inhibits the transmembrane influx of calcium ions into vascular smooth muscle and cardiac muscle cells and is used in the management of hypertension and angina pectoris.3 Like most other calcium blocker agents of the dihydropyridine type, AM is therapeutically used as the racemic mixture of R(+) and S( ) enantiomers. However, the S( ) enantiomer has shown to be more than 2000 times more potent than the R(+) enantiomer in calcium channel blocking activity.4,5 As a result of different pharmacologic and toxicologic properties of enantiomers, enantioselective analytical and preparative methods are required not only for pharmacodynamic and pharmacokinetic studies, but also for quality control of pharmaceutical preparations and determination of their enantiomeric purity.6 Chromatography and electromigration techniques have long been the methods of choice for enantioseparation analysis.7 In the last years, capillary electrophoresis (CE) has been increasingly applied for the enantioseparation of pharmaceutical compounds, due to its several attractive features such as speed of method development and analysis, robustness, simplicity, and low cost. Typical enantiomer separations by CE are performed by simply applying chiral additives to the running buffer of CE. Polysaccharides, proteins, crown ethers, and chiral surfactants are some of the chiral additives that have been used for CE enantiomer separation studies.8 Recently, liquid chromatography-mass spectrometry (LC-MS) was applied for quantification of AM enantiomers in human plasma.9 Also, AM enantiomers were separated on a molecularly imprinted polymer-based stationary phase.10 In another work, chiral LC was used for determination of enantiomeric purity of S( )-AM.11 AM enantiomers have been separated by a number © 2014 Wiley Periodicals, Inc.
of CE methods, most of them using cyclodextrins (CDs) as chiral selectors. Five neutral CDs (α-CD, β-CD, γ-CD, hydroxypropyl-βCD [HP-β-CD] and hydroxyethyl-β-CD) and the anionic sulphobutylether-β-CD and carboxymethyl-β-CD have been examined for the resolution of AM enantiomers in both highperformance liquid chromatography (HPLC) and CE.12 In different reports, hydroxypropyl-α-CD (HP-α-CD) and HP-β-CD modified CE procedures were used for quantification of AM enantiomers in commercial tablets, human plasma, and serum samples.2,13–16 Also, polybrene, a cationic polymer, was used for improvement of AM enantioseparation using CE.17 An on-line coupled isotachophoresis-capillary zone electrophoresis (CZE) separation method using HP-β-CD as chiral selector enabled the direct detection and quantitation of AM enantiomers in urine samples.18 Dual chiral selector systems consisting of glycogen (neutral polysaccharide) with chondroitin sulfate A (ionic polysaccharide), β-CD, and HP-β-CD exhibited good enantioselective properties for AM analysis.19 Recently, polysaccharide-based chiral columns were applied for enantiomer separation of dihydropyridine derivatives.20 In another work, enantiomeric separation of AM and its two chiral impurities by nano-LC and capillary electrochromatography (CEC) using a chiral stationary phase based on cellulose tris(4-chloro-3methylphenylcarbamate) was investigated.21 While CDs are cyclic oligomers, produced from starch by means of enzymatic conversion, maltodextrins and heparin are other polysaccharide-based compounds that have been *Correspondence to: Ali Mohammadi or Saeed Nojavan, Department of Drug & Food Control, Faculty of Pharmacy, Tehran University of Medical Sciences, P.O. Box 14155–6451, Tehran 14174, Iran and Faculty of Chemistry, Shahid Beheshti University, G. C., P.O. Box 19396–4716, Evin, Tehran, Iran. E-mail: [email protected] or [email protected] Received for publication 9 January 2014; Accepted 28 March 2014 DOI: 10.1002/chir.22334 Published online 16 May 2014 in Wiley Online Library (wileyonlinelibrary.com).
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All solutions were prepared by HPLC-grade water that was obtained from a Milli-Q system (Millipore Milford, MA). Buffer and all solutions were filtered through 0.45-μm PTFE membrane filters before use.
CE Equipment Fig. 1. Chemical structure of AM. The asterisk denotes the chiral center.
investigated for their chiral selector properties. Maltodextrins are complex malto-oligo and polysaccharide mixtures obtained from partial acid and/or enzymatic hydrolysis of starch. They are characterized by their dextrose equivalent (DE), which is the equivalent of the degree of polymerization of malto-oligosaccharides. The higher the DE number, the higher is the extent of starch hydrolysis and, consequently, the shorter are the oligomeric chains present in a mixture. Different interactions of chiral solutes with the helical structure of the maltodextrin emerge as the basis of the enantioselectivity. When used in CE, maltodextrins have shown highly efficient chiral selectivity for a broad range of acidic and basic compounds. They were used in the design of potentiometric, enantioselective membrane electrodes for analysis of captopril and baclofen.22,23 Chiral separation of cetirizine and pentazocine was achieved by CE methods using maltodextrins.24,25 Maltodextrin was also used for stability evaluation of tramadol enantiomers using a chiral stability indicating CE method.26 Heparin is a glycosaminoglycan, a heterogeneous mixture of variably sulfated polysaccharide chains. The large but variable degree of sulfation results in heparin being one of the most anionic biopolymers. The highly anionic character of heparin enhances its solubility, while offering the potential for considerable electrophoretic mobility. The electrophoretic mobility of heparin, coupled with its helicity in conjunction with the chirality inherent in the constituent monosaccharide residues suggested it as a chiral mobile phase additive in CE analysis and was successfully used for CE enantioseparation of several antimalarial drugs, antihistamines, and other pharmaceutical compounds.27,28 In the present study, we evaluated the applicability of maltodextrins with different DE values and heparin in order to develop a chiral CE analysis method for AM in commercial tablets. Other contributing factors such as the chiral selector concentration, pH of background electrolyte (BGE), BGE concentration, voltage, and cartridge temperature were optimized and the method was validated and proved to be precise, accurate, and enantioselective enough to be used for quality control of AM enantiomers in tablet dosage form. Using maltodextrin as a commercial chiral selector made this method an inexpensive alternative for previous CE methods, which are usually based on CDs or noncommercial chiral selectors. EXPERIMENTAL Reagents and Materials AM besylate racemic powder and its R(+) and S( ) enantiomers (purity >99.90%) were obtained from Tofigh Daru Research & Engineering Company (Tehran, Iran). AM besylate tablets (Amlotidi 5 mg) made by Tolidaru Pharmaceutical Company (Tehran, Iran) were used for assay studies. Maltodextrins with three different DE values (4–7, 13–17, and 16.5–19.5) were purchased from Sigma-Aldrich Chemie (Steinheim, Germany). Hydrochloric acid, sodium hydroxide, phosphoric acid, Na2HPO4, and NaH2PO4.2H2O salts were purchased from Merck (Darmstadt, Germany). All chemicals and reagents were of analytical grade and were used as received.
CE experiments were carried out using a Lumex Capel 105 (Lumex, St. Petersburg, Russia) equipped with a UV detector, set at 214 nm and an uncoated fused-silica capillary (Lumex), 57 cm × 75 μm I.D. (49 cm effective length). The cartridge temperature was maintained at the desired amount by a water-circulating system. Capillary conditioning was performed daily before the first experiment as follows: 20 min with 0.5 M HCl, 5 min with water, 30 min with 0.5 M NaOH, and 5 min with water. Additionally, the capillary was washed for 2 min with water, 2 min with 0.5 M NaOH, 2 min with water, and 2 min with the running buffer with positive pressure applied at the injection end before each run. Acquisition of electropherograms was computer-controlled by Chrom&Spec software v. 1.5. The analytes were injected at the anodic end by applying the pressure of 60 mbar for 6 s.
Preparation of Stock and Standard Solutions A stock solution of racemic AM (1000 μg/mL) was prepared in water. Stock solutions were protected from light and stored for 3 weeks at 4°C without any decomposition. Aliquots of AM standard stock solution were transferred into 100-mL volumetric flasks and solutions were made up to volume with water to yield final concentrations of 1, 2.5, 5, 10, 25, 50, 100, 150, 200, and 250 μg/mL for each enantiomer.
Preparation of Tablets for Assay Twenty AM tablets were weighed, crushed, and mixed in a mortar and pastel for 20 min. A portion of powder equivalent to the weight of one tablet was accurately weighed and transferred into a 25-mL A-grade volumetric flask and 5 mL water was added to the flask. The volumetric flask was sonicated for 20 min until complete dissolution of the drug. The solution was then made up to volume with HPLC-grade water. An aliquot of the solution was filtered through a 0.45-μm nylon filter and was transferred to a 10-mL A-grade volumetric flask and made up to volume with water to yield a concentration of each enantiomer in the range of linearity.
RESULTS AND DISCUSSION Method Development and Optimization Using Maltodextrin Effect of BGE pH on the enantioseparation. The BGE pH is one
of the most important parameters in CE analysis because of its effect on the electroosmotic flow (EOF) and ionization state of the analyte.29 The AM chemical structure consists of a free amino group with pKa 8.6, which means the molecule is positively charged at pH values under 8.6 and neutral at higher pH values.30 Therefore, it can be deduced that AM is electrophoretically separable at neutral and acidic pH values by normal phase CE methods. EOF is at its lowest amount at pH values below 2 and increases gradually with an increase in pH, which results in shorter migration times and decreased resolution. Different pH values in the range of 2–5 were investigated in order to obtain the optimum amount based on the enantioresolution and rational migration time (Table 1). Although the two enantiomers were best resolved at pH 2, pH 4 was chosen as the optimum pH for further experiments because shorter migration times were obtained for enantiomers and the resolution was not affected significantly. The migration order of each enantiomer was identified using the pure R(+) or S( )-enantiomer. The results showed that R(+)-enantiomer migrated faster than the S( )-enantiomer. Effect of maltodextrin DE value on the enantioseparation. Malto-
dextrins with different DE values (4–7, 13–17, and 16.5–19.5) were evaluated for their chiral selector activity and, as shown Chirality DOI 10.1002/chir
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TABLE 1. Effect of BGE pH on the migration times and resoa lution of AM enantiomers
sites to interact with the analyte molecules which can explain the observed results.24
pH
Effect of maltodextrin concentration on the enantioseparation. Malto-
2 3 4 5
t(R+) (min)
t(S-) (min)
Resolution
34.18 29.88 24.78 20.82
35.01 30.68 25.17 21.18
1.61 1.59 1.45 1.39
a
Experimental condition: capillary column: 57 cm (49 cm effective length), 75 μm id; UV detection: 214 nm; applied voltage: 20 kV; temperature: 25 °C; injection: 6 s (at a pressure of 60 mbar); concentration of each enantiomer: 25 μg/mL, BGE: 100 mM phosphate buffer, containing 10% w/v maltodextrin (DE value 4–7).
in Fig. 2, maltodextrin with DE value 4–7 separated the enantiomers with better resolution and longer migration times. Since maltodextrin is a neutral polysaccharide, hydrophobic and hydrogen bonds are the most important interactions responsible for separating the enantiomers and maltodextrins with lower DE values and longer oligomeric chains have more
dextrin was added to the phosphate buffer at four different concentrations (5, 10, 15, and 20% w/v) and its effects on the migration times and resolution of AM enantiomers were investigated. It was observed that increasing the maltodextrin concentration induced improvement of enantioresolution through the elongation of the migration times. Higher maltodextrin concentrations introduced more interaction sites for AM molecules that resulted in better separation of the enantiomers. Also, increasing of maltodextrin concentration increased the BGE viscosity, which was another reason for the elongated migration times. This effect also prevented us from increasing the maltodextrin concentration above 20% w/v. In order to obtain rational resolution and migration times, 10% w/v was chosen as the optimum maltodextrin concentration (Fig. 3A). Effect of BGE concentration on the enantioseparation. Phosphate
buffer was investigated at four different concentrations; (50, 75, 100, and 120 mM). This parameter affects the migration time by decreasing the EOF and increasing the viscosity and improves the resolution by providing better interactions between AM molecules and maltodextrin through inhibiting their adsorption on the capillary interior wall. According to the data presented in Table 2 and to maintain a balance between resolution and migration time, 100 mM was chosen as the optimum concentration for the phosphate buffer. Effect of applied voltage on the enantioseparation. The effect of
applied voltage on chiral CE analysis can be discussed from different aspects. Increased amounts of voltage usually improve the CE analysis efficiency, decrease the migration times by affecting both EOF and electrophoretic flow, and have a
Fig. 2. Effect of maltodextrin DE value on the migration times and resolution of AM enantiomers: (A) DE value 16.5–19.5; (B) DE value 13–17 and (C) DE value 4–7. Experimental conditions: capillary column: 57 cm (49 cm effective length), 75 μm id; UV detection: 214 nm; applied voltage: 20 kV; temperature: 25 °C; injection: 6 s (at a pressure of 60 mbar); concentration of each enantiomer: 25 μg/mL, BGE: 100 mM phosphate buffer, pH 4.0 containing 10% w/v maltodextrin. Chirality DOI 10.1002/chir
Fig. 3. Effect of (A) maltodextrin concentration and (B) applied voltage on the enantioseparation of AM enantiomers. Experimental conditions: capillary column: 57 cm (49 cm effective length), 75 μm id; UV detection: 214 nm; applied voltage: 20 kV; temperature: 25 °C; injection: 6 s (at a pressure of 60 mbar); concentration of each enantiomer: 25 μg/mL, BGE: 100 mM phosphate buffer, pH 4.0, DE value of maltodextrin: 4–7. In (B) the concentration of maltodextrin was 10% w/v.
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DETERMINATION OF AMLODIPINE ENANTIOMERS
TABLE 2. Effect of buffer concentration on resolution of a amlodipine enantiomers Phosphate buffer concentration (mM)
t(R+) (min)
t(S-) (min)
Resolution
20.23 22.47 24.78 27.63
20.43 22.75 25.17 28.12
0.92 1.1 1.45 1.54
50 75 100 120 a
Experimental condition: capillary column: 57 cm (49 cm effective length), 75 μm id; UV detection: 214 nm; applied voltage: 20 kV; temperature: 25 °C; injection: 6 s (at a pressure of 60 mbar); concentration of each enantiomer: 25 μg/mL, BGE: phosphate buffer, pH 4.0 containing 10% maltodextrin (DE value 4–7).
significant role in Joule heating through increasing the electric current. Voltage was investigated in the range of 12–22 kV in order to reach an optimum amount for separating AM enantiomers. Decreasing the voltage from 22 to 15 kV increased the resolution probably because of decreasing the migration times and providing enough time for interaction of AM and maltodextrin molecules. However, further reduction in voltage from 15 to 12 kV decreased the resolution, which shows the importance of voltage in CE analysis efficiency. Thus, an applied voltage of 20 kV was chosen as the optimum voltage in order to obtain a good resolution with reasonable migration times (Fig. 3B). Effect of cartridge temperature on the enantioseparation. The car-
tridge temperature can affect the EOF and electrophoretic flow through changing the BGE viscosity, which also explains its effect on the resolution. Several temperatures in the range of 15–30 °C were examined in order to find the optimum temperature at which there is a good resolution and reasonable migration times. Increasing the temperature from 15–30 °C, decreased the migration times and resolution due to shorter time for essential interactions. Although the best resolution was achieved at 15 °C, the temperature of 20 °C was chosen as the optimum temperature because of short migration time of each enantiomer in this temperature (Table 3). Method Development and Optimization Using Heparin Evaluation of enantioseparation capability of heparin. Previous
studies have shown that enantioseparation using heparin as a chiral selector can be performed in either normal or reversed polarity.27,28 In this study, at normal polarity, adding heparin into the BGE with pH values in the range of 2–8 did not result in any enantioseparation.
TABLE 3. Effect of cartridge temperature on the migration a times and resolution of amlodipine enantiomers Temperature (°C) 15 20 25 30 a
t(R+) (min)
t(S-) (min)
Resolution
31.17 25.71 22.91 18.18
31.65 26.19 23.26 18.49
1.94 1.68 1.45 1.30
Experimental condition: capillary column: 57 cm (49 cm effective length), 75 μm id; UV detection: 214 nm; voltage: 20 kV; injection: 6 s (at a pressure of 60 mbar); concentration of each enantiomer: 25 μg/mL, BGE: 100 mM phosphate buffer, pH 4.0 containing 10% w/v maltodextrin (DE value 4–7).
Enantioseparation by heparin has also been performed at reverse polarity.28 This experiment was performed by adding heparin into the BGE pH 2. AM was not detected in the resultant electropherogram. This observation together with the normal CE analysis results indicated that there are no interactions between AM and heparin molecules. Stalcup and Agyei,27 who investigated the enantioseparation of several compounds using heparin, concluded that the analyte molecular structure and size are important factors in heparin recognition ability. It seems that the AM molecule does not have the required criteria to produce efficient interactions with heparin. Higher heparin concentrations up to 6% w/v were also investigated to see whether they can make the desired interactions with AM molecules. Increasing the heparin concentration did not have any effect on the enantioseparation but increased the migration times, probably because of the BGE viscosity. Effect of heparin on the enantioseparation in the presence of other chiral selectors. Heparin was added to the BGE containing
maltodextrin or HP-α-CD, and their overall enantioseparation activity was investigated. Maltodextrin and HP-α-CD could resolve AM enantiomers; however, adding 1% w/v heparin deteriorated their chiral selector activity. These results showed that heparin was not only unable to separate AM enantiomers, but also decreased the efficiency of other chiral selector compounds by blocking their interaction sites. Validation of the Method
After choosing maltodextrin as the optimum chiral selector and optimizing various parameters that affect its efficiency, the method was validated for linearity, limit of detection (LOD), limit of quantitation (LOQ), precision, accuracy, and recovery. Linearity. Plots of peak area (Y) against concentration
(X, μg/mL) were drawn for both AM enantiomers. Both plots were linear in the range of 2.5–250 μg/mL and regression equations were calculated as Y = 0.80 X + 3.92 for R(+)-enantiomer and Y = 0.88 X + 3.32 for S( )-enantiomer. Correlation coefficients (R2) were 0.9991 and 0.9992 for R(+) and S( )enantiomers, respectively. LOD and LOQ. According to the pharmacopeial recommen-
dations (United States Pharmacopeia, 2013), the LOD and LOQ were determined for both enantiomers, at signal-tonoise ratios of 3:1 and 10:1, respectively. The LOQ value (1.7 μg/mL) was verified by its nearness to the lower concentration of the working range. The LOD (0.52 μg/mL) was then calculated by dividing LOQ by 3.3. Interday precision was assessed by preparing standard samples with 25 and 250 μg/mL concentrations for each enantiomer and analyzing them in three different days. Intraday precision was assessed by analyzing the above-mentioned standard samples five times in a single day. Relative standard deviations (RSD) are presented in Table 4. We also calculated each enantiomer concentration and relative error (RE) using the average peak areas and the regression equations, which show the method accuracy.
Precision and accuracy.
Recovery. The recovery test was performed in order to inves-
tigate the possible interactions of tablet matrix with analysis of the drug substance. A known amount of standard powder was added to the sample of tablet powder and diluted to yield a concentration of 200 μg/mL for each enantiomer. This sample was analyzed using the optimized method five times. Chirality DOI 10.1002/chir
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TABLE 4. Intra- and interday precision and accuracy of AM enantiomers Found concentration (μg/mL), RSD (%), RE (%) Enantiomer
Actual concentration (μg/mL)
R-(+)
25 250 25 250
S-( )
Intraday 25.47, 249.06, 25.18, 248.85,
Interday
5.1, +1.88 1.8, 0.38 6.1, +0.72 1.9, 0.46
25.55, 5.1, +2.2 247.71, 1.9, 0.92 25.66, 7, +2.64 248.4, 2.1, 0.64
LITERATURE CITED
Fig. 4. Assay of AM R(+) and S( )-enantiomers in a commercial tablet. Experimental conditions: capillary column: 57 cm (49 cm effective length), 75 μm id; UV detection: 214 nm; temperature: 20 °C; voltage: 20 kV; injection: 6 s (at a pressure of 60 mbar); BGE: 100 mM phosphate buffer pH 2 containing 10% w/v maltodextrin (DE value 4–7).
The observed concentrations of R(+) and S( )-enantiomers were found to be 194.45 ± 3.56 μg/mL (mean ± SD) and 195.57 ± 3.81 μg/mL, respectively. The resultant % RSD of these studies were found to be 1.83 % for R(+)-enantiomer and 1.95 % for S( )-enantiomer with the corresponding recovery values of 97.2 % and 97.8 %, respectively. These results indicated that the method was selective for the analysis of both enantiomers without any interference from the excipients used in the formulation and production of the tablets. Assay of AM enantiomers in tablets. The optimized and vali-
dated CE method was used for analysis of AM enantiomers in commercial AM tablets. The amounts of AM R(+) and S ( )-enantiomers were found to be 102.5% (% RSD = 2.1) and 109.3% (% RSD = 5.2) of the label claimed amount through five analyses. The actual amounts are therefore 2.56 mg for R(+)enantiomer and 2.73 mg for S( )-enantiomer. Fig. 4 shows one of the assay electropherograms. CONCLUSIONS
Maltodextrin, a neutral polysaccharide, as a chiral selector was investigated for separation of AM enantiomers. All maltodextrins with different DE values separated AM enantiomers, while the one with DE value 4–7 showed the best results and was used for development and validation of a stereoselective CE method for determination of AM enantiomers in commercial tablets. The method was optimized for its various parameters and produced good resolution between AM enantiomer peaks in a reasonable time. Also, enantioseparation capability of heparin was investigated and the results showed that heparin did not have any chiral selector activity in this study. ACKNOWLEDGMENT
The authors acknowledge the financial assistance from Tehran University of Medical Sciences, Tehran, Iran. The authors thank Shahid Beheshti University for the supply of analytical instruments. Chirality DOI 10.1002/chir
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Chirality DOI 10.1002/chir
