Enantiomeric analysis of rivastigmine in pharmaceuticals by cyclodextrin-modified capillary zone electrophoresis

Analytica Chimica Acta 525 (2004) 43–51

Enantiomeric analysis of rivastigmine in pharmaceuticals by cyclodextrin-modified capillary zone electrophoresis Andrea Kaval´ırov´aa,b,∗ , Marie Posp´ısˇilov´ab , Rolf Karl´ıcˇ ekb b

a Faculty of Pharmacy, The Research Centre LN00B125, Hradec Kr´ alov´e, Czech Republic Department of Analytical Chemistry, Faculty of Pharmacy, Charles University, Heyrovsk´eho 1203, CZ-50005 Hradec Kr´alov´e, Czech Republic

Received 22 March 2004; received in revised form 10 August 2004; accepted 10 August 2004 Available online 3 September 2004

Abstract Chiral separation method development is usually very time-consuming due to the diversity in chemical structures of pharmaceutical drug substances as well as the suitable separation conditions and the problem to choose the appropriate chiral selector. This paper shows capillary zone electrophoresis (CZE) which was developed for chiral separation of a basic compound – rivastigmine (RIV) using 30 cm × 50 ␮m i.d. polyacrylamide (PAA)-coated fused-silica capillary (effective length 20 cm), amine-modified phosphate buffer of pH 2.5 and sulfated-␤-CD (S-␤-CD) as chiral selector. Other selected native or derivatized cyclodextrins (CDs) were also tested: ␤-CD (5, 30 mM), carboxymethyl␤-CD (5, 30 mM), dimethyl-␤-CD (15 mM), hydroxypropyl-␤-CD (5, 30 mM), hydroxypropyl-␣-CD (5, 30 mM) and hydroxypropyl-␥-CD (5, 30 mM). Complete enantiomeric separation of RIV was achieved at 20 kV, 18 ◦ C and detection at 200 nm within 8 min with R.S.D. for the absolute migration time reproducibility of less than 2.1%. Rectilinear calibration range was 5.0–500.0 ␮M of each enantiomer (r = 0.9994–0.9995). The CZE method proposed was used for the control of chiral purity of pharmaceutically active S-RIV and for the analysis of Exelon caps preparation. © 2004 Elsevier B.V. All rights reserved. Keywords: Capillary zone electrophoresis; Enantiomer separation; ␤-Cyclodextrin derivatives; Rivastigmine

1. Introduction Usually one of the enantiomers of a racemic drug exhibits desirable therapeutic activity or the individual enantiomers can possess quite different pharmacological properties and toxicity. Drug regulatory agencies in many countries have expressed an interest in investigations of the stereoisomeric composition of drugs and their associated therapeutic and toxicological consequences. In general, only highly biologically active enantiomers are used in the production of drugs [1]. For that reason the safety of chiral pharmaceuticals is critically related to their chiral purity control. Analytical methodology is required to assess the presence of impurities in chiral

∗

Corresponding author. Tel.: +42 495067265; fax: +42 495518718. E-mail address: [email protected] (A. Kaval´ırov´a).

0003-2670/$ – see front matter © 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.aca.2004.08.026

drugs during various stages of manufacturing of these pharmaceuticals. Chiral gas (GC) and liquid chromatography (HPLC) were the first tools employed in the analytical separation of enantiomers [2]. In the last several years, CZE has become a powerful technique as an alternative to chiral chromatography methods. CZE with high resolving power and intrinsic selectivity is predestined to play an important role in chiral drug separation and offers some important advantages such as simplicity, short analysis time, flexibility and consumption of relatively small volumes of samples and buffers. The separation principle of CZE is based on the different electrophoretic mobilities of solutes, which in turn, depend on their charge densities [3]. Similar to HPLC, secondary equilibrium also can be employed in CZE separations by means of the use of special additives in the running buffer, such as inclusion complex agents like cyclodextrins (CDs). CD inclu-

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sion complexes can be used for the optical resolution of enantiomers by CZE, based on the differences in electrophoretic mobility of the complexes arising from different complexformation constants with the analyte [4]. The applications of CZE-chiral techniques have been extensively described in recent publications [5–7]. Various types of chiral selectors, such as crown ethers [8,9], CDs [10–12], proteins [13–15], antibiotics [16,17] and polysaccharides [18] have been used for separation of enantiomers either by adding them into the CZE electrolyte buffers or by immobilizing onto the internal surface of the capillary wall [19]. Rivastigmine (S)-N-ethyl-3-[(1-dimethyl-amino)ethyl]N-methyl-phenylcarbamate hydrogen tartrate, is a reversible and non-competetive acethylcholinesterase inhibitor of the second generation, selectively inhibiting its activity in certain brain areas. The structure of RIV is shown in Fig. 1. The compound also enhances cholinacetyltransferase activity that stimulates acetylcholine synthesis. RIV as a carbamate derivative of physostigmine proved successful in the treatment of Alzheimer’s disease. It was originally synthesized as a racemate but later developed and marketed as the more potent S-(−)-enantiomer. The concentration of R-enantiomer required to reduce acetylcholinesterase activity is 10-fold that of S-enantiomer. Gas chromatography–mass spectrometry (GC–MS) and liquid chromatography with atmospheric pressure chemical ionization tandem mass spectrometry (LC–MS–MS) was developed for simultaneous determining of RIV and its major metabolite in human plasma with the limit of quantitation of 0.2 ng ml−1 [20,21]. Rawjee and Vigh [22] showed that basic compounds could be involved in three different types of CD-modified CZE separations. The separation is desionoselective when only the non-dissociated enantiomer complexes selectively with the CD. The separation is ionoselective when only the dissociated enantiomer complexes selectively, and the separation is duoselective when both the non-dissociated and dissociated enantiomer complex selectively with the chiral selector. Chiral separation method development of basic compounds should be primarily based on the change of the pH of the running buffer, CD type and concentration. Once the initial separation conditions have been achieved (pH, CD type, CD concentration), further enhancements can be made by adjusting the applied electric field strength, temperature and capillary length in order to decrease the analysis time. The use of CDs for the separation of enantiomers of basic drugs has been reported formerly [23,24]. Mikus et al. has been demonstrated high resolving power of charged CD derivatives as an alternative to native CDs [25].

Fig. 1. Structure of rivastigmine hydrogen tartrate.

Selection of the appropriate chiral selectors for a given chiral compound is not straightforward since predictions of the enantioselectivity of CDs based on the molecular structures of guest molecules are often difficult. Therefore, it is necessary to evaluate the different enantioselectivity of CDs for a wide range of chiral compounds in order to understand the chiral interaction between a guest molecule and a cavity of CD. This paper describes a simple, cost-effective and reliable method for enantiomeric separation of basic drug, namely RIV, using several CDs including neutral and charged CD derivatives. To gain further insight into the stereospecific interactions between S-␤-CD and RIV, the effect of the molecular structure of RIV on its migration behaviour was also investigated. CZE with negatively charged CDs was expected to be more effective than the CZE with a native ␤-CD. Furthermore, the method proposed was applied for the control of the purity of the active enantiomer in bulk drug. To our best knowledge, the enantiomeric separation of the RIV has not yet been presented in existing literature.

2. Experimental 2.1. Apparatus and CZE procedure All experiments were performed using a P/ACE MDQ system equipped with a diode array detection system (Beckman Coulter, Fullerton, CA, USA). The temperature of the cartridge holding the capillary column was maintained at 18 ± 0.1 ◦ C by the liquid cooling system of the P/ACE instrument. The separations were monitored at 200 nm. The system was computer-controlled, with an integrated P/ACE Station Software (32 KaratTM Version 4.0) package. Two different types of capillaries were utilized: a bare silica capillary of 60 cm × 50 ␮m i.d. or a PAA-coated capillary of 30 cm × 50 ␮m i.d. with the detection window at the distance of 10 cm from the capillary outlet. Running procedure: a new fused-silica capillary was flushed successively with 1 M NaOH (30 min), 0.1 M NaOH (10 min) and deionised water (40 min). Before use, the capillary was rinsed with 1 M NaOH (5 min), 0.1 M NaOH (5 min), water (10 min) and running buffer (5 min). The conditioning between measurements was effective by rinsing with 0.1 M NaOH (3 min), water (3 min) and running buffer (2 min). Before each analysis, a PAA capillary was rinsed successively with 0.1 M HCl (0.5 min), deionised water (2 min), and the running buffer (2 min). This procedure was also used when a new PAA capillary was employed. The injection mode was hydrodynamic (4 s, 7 mbar pressure). When using a longer capillary, 30 kV was applied across a 50 ␮m i.d. × 60 cm bare silica capillary with normal polarity (detection at cathode). When using a shorter capillary, 20 kV was applied across a 50 ␮m i.d. × 30 cm PAA-coated capillary with normal polarity too.

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2.2. Chemicals

3.1. Screening method development

Chiral selectors: ␤-CD, sulfated-␤-CD (S-␤-CD, with nominal degree of substitution ∼7–11), carboxymethyl-␤CD (CM-␤-CD), phosphoric acid and tris[hydroxymethyl]aminomethane (TRIS) were obtained from Sigma (St. Louis, MO, USA). Dimethyl-␤-CD (DM-␤-CD), carboxymethyl-␤-CD (CM-␤-CD), hydroxypropyl-␣-CD (HP-␣-CD), hydroxypropyl-␤-CD (HP-␤-CD), hydroxypropyl-␥-CD (HP-␥-CD) were supplied by Fluka (Steinheim, Switzerland). Triethylamine (TEA) was obtained from Sigma (Prague, Czech Republic). Samples: R-(+) and S-(−)-RIV hydrogen tartrate were kindly donated by the Institute for the Control of Pharmaceutical and Biological Products, Prague, Czech Republic. Other chemicals were of analytical grade.

3.1.1. General consideration Resolution, RS , between two enantiomers in CZE in the presence of electroosmosis can be expressed as [26]: √ N µ RS = (1) 4 µAV + µeo

2.3. Sample and buffer preparation

(a) Enhancing enantioselectivity: After the understanding of the chiral interactions between the chiral selector (CD) and the solute molecules, the choice of a suitable chiral agent is very important. RS could be improved by optimising the concentration of CD since RS is a function of [CD]. However, since RS depends on the formation constants of inclusion complex (KCD1 and KCD2 ) the optimal concentration of CD may be different from one pair of chiral compounds to another. (b) Controlling electroosmosis: Maximum RS could be obtained when µeo = −µAV at the expense of analysis time approaching infinity. Methods using coated capillary columns at low pH conditions are intended to eliminate or reduce the EOF. In a bare fused-silica capillary, the migration of basic compounds is in the same direction of the EOF. Therefore, it is possible to increase enantiomeric resolution by reversing the EOF to counteract the migration of the basic enantiomers. The main drawback is the loss of efficiency due to longitudinal diffusion as the migration times of solutes are prolonged.

The racemic test mixtures of both enantiomers (R/S = 1) were stored at −20 ◦ C or used freshly prepared to prevent degradation. Exelon® capsules (Novartis, Basel, Switzerland) were analysed. Standard stock solutions of R- and SRIV with concentration of 1100 ␮g ml−1 were prepared in methanol–buffer (1:4); deionised water was used for further dilution. The running buffer was prepared by dissolving exact amount of S-␤-CD in an appropriate volume of 100 mM phosphoric acid. Its pH was adjusted to 2.5 by using TRIS, TEA or NaOH. Ultrapure water prepared with a Millipore Water Purification System (Watford, UK) was used throughout. The electrolyte solutions were filtered through 0.45 ␮m membrane filters (PuradiscTM 45PP, Whatman, New Jersey, USA). pH meter (PHM 220, Radiometer Copenhagen) equipped with pHC2401-8 combined glass electrode was used for pH measurements.

where N is the number of the theoretical plates, µeo the electroosmotic mobility, µAV the average electrophoretic mobility and µ the mobility difference for a pair of enantiomers. Baseline separation is generally obtained when RS exceeds 1.5. There are several methods for improving enantiomeric resolution, RS :

2.4. Analysis of Exelon (capsules) The content of 10 capsules was emptied and homogenised. The weight of the powder equivalent to ∼160 mg of RIV was transferred into a 100 ml volumetric flask with 60 ml of methanol and the flask was packed. The suspension was kept in an ultrasonic bath for 10 min and then centrifuged at 1300 × g for 15 min. About 5.00 ml of the supernatant was diluted to 10 ml in a volumetric flask and the solution was subjected to a CZE analysis.

3. Results and discussion The migration behaviour of RIV in a bare fused-silica and a PAA capillary was investigated. The quality of chiral separation should be improved by modifying the inner surface of the capillary. Some elaborated methods have been used coated capillaries to minimize adsorption of analyte onto the capillary wall and to reduce the electroosmotic flow.

A low pH buffer has been selected to enhance solubility and to generate mobility by protonation of the amine-buffers. Sample mobility is necessary for use with the neutral CD buffers. The purpose of adding TRIS or TEA to the buffers is suppress the EOF in order to increase the enantioselectivity, and to eliminate analyte interactions with the uncoated capillary wall [27]. Typical electrophoretic mobility of basic compounds is ∼1 × 10−4 cm2 V−1 s−1 for the amine–phosphate buffers with neutral CDs. Therefore, basic compounds migrate toward the cathode even though the EOF is slightly reversed. The TEA–phosphate buffer used with the S-␤-CD had a weak, cathodic EOF of ∼1 × 10−5 cm2 V−1 s−1 [28]. Compared to neutral capillaries, under these conditions in a bare silica capillary achieved the same goal of suppressing EOF, while being more rugged and economical. Quang and Khaledi [29] have shown that the use of shortchain tetraalkylammonium (TAA) cations in acidic (pH 2.5) buffer solutions leads to a reversal in the direction of the EOF. The enantiomeric resolution of basic compounds can be im-

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proved as the EOF counteracts the migration of enantiomers. The advantage of the short-chain TAA cations as compared to long-chain cationic surfactants is that the former ones do not interact strongly with CDs, leaving the chiral recognition sites more accessible for solute enantiomers. On the other hand, the short-chain TAA cations effectively cover the fused-silica surface, leading to a reversal of the EOF at the acidic condition of pH 2.5. The use of a coated low-EOF capillary also has been ensured the predicted migration direction of the basic RIV with the low-pH running buffer at normal polarity (anode at the injection side). The baseline was improved slightly and significantly stabilized when a PAA-coated capillary was used instead of a fused silica, due to the marked reduction of current. The capillary coating also has been reduced any peak tailing. 3.2. The electrolyte system – the buffer type, concentration and pH The most important step in method development of chiral separations is the choice of an adequate type of chiral selector and its concentration. Optimisation of a number of other experimental conditions, such as the buffer type and its concentration, pH, content of various modifiers, separation temperature and voltage, contributes to the highest degree of separation. Fig. 2A and B indicates that racemic RIV has been separated into its enantiomeric isomers with typical resolution that has been much greater than unity within 8 min (depend-

Fig. 2. Chiral separation of rac-RIV; phosphate/TRIS 100 mM, pH 2.5 containing S-␤-CD (7 mM); applied voltage, +25 kV (A), +30 kV (B); PAAcoated capillary, 50 ␮m i.d. with 20 cm effective length (A), 50 cm (B); λ = 200 nm.

Fig. 3. Chiral separation of RIV in phosphate/TRIS 100 mM (A) and phosphate/TEA 100 mM (B), pH 2.5 (7 mM S-␤-CD, +20 kV, PAA-effective length 20 cm/50 ␮m i.d.).

ing on the effective length of capillary) under the optimised conditions (pH 2.5; content of chiral selector 7 mM; buffer concentration 100 mM; temperature 18 ± 0.1 ◦ C with PAAcoated capillary). In this work, sodium, TRIS and TEA were used as counterions in phosphate buffer of pH 2.5 and their effect on separation selectivity was compared. Although the EOF was reversed under buffer conditions (Fig. 2A) the magnitude of µeo was still smaller than that of the mobility of RIV. Therefore RIV migrates still towards the negative electrode. A distinct difference was observed when using TEA and TRIS as buffer cations (see Fig. 3A and B). A complete chiral separation of RIV was achieved when using 100 mM phosphate buffer (pH 2.5) containing S-␤-CD. As compared to the TRIS cations, the TEA cations have many significant features such as providing different separation selectivity, increasing the solubility of ␤-CD [26]. It was found that the TEA cations compete with analyte to some extent for the hydrophobic cavity of ␤-CDs, which reduces the enantiomeric resolution of RIV. In this regard, the TRIS cations are less competitive due to a shorter alkyl chain, and generally more suitable for chiral separation of basic RIV. The chiral separations were performed in TRIS–phosphate buffer with S-␤-CD as chiral selector. The effect of the concentration of the phosphate in the buffer solution on the migration time of R/S-RIV is demonstrated in Fig. 4. The influence of buffer concentration on the generation of electric current was tested. The values of generated current were increased in increasing of buffer concentration. The baseline separations were achieved with 90–110 mM phosphate of pH 2.5 and 7 mM of S-␤-CD. In order to prevent the generation

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allowed for interactions between the used CD and the analyte. Also, the enantioseparation mechanism(s) involved include the formation of inclusion complexes which could be unstable at pH greater than 4.5. Furthermore, the drug molecule protonation degree is smaller at higher pH which may cause better fitting of the analyte molecule into the S-CD cavity of the chiral selector resulting in faster migration of the negatively charged complex formed. 3.3. Effect of the type and concentration of CDs

Fig. 4. Effects of phosphate/TRIS buffer concentrations on the migration time of rac-RIV; (
) S-RIV; () R-RIV. CZE conditions are the same as in Fig. 3.

of extensive Joule heat the 100 mM phosphate buffer was chosen as optimal. Effect of the buffer pH on the migration times of the drug embraces a number of aspects such as the degree of protonation of the nitrogen atom of the drug, compatibility of the protonated drug molecule with the lipophilic CD cavity of the chiral selector, and the electrostatic interactions among the analyte, the chiral selector and the buffer component TRIS at high concentrations. The effect of various pH values (2.5, 3.0, 4.0, 5.5, 6.5 and 7.5) on the resolution (RS ) of R/S-RIV in running buffer with 7 mM S-␤-CD is presented in Fig. 5. RIV has pK value of about 8.6 [30] and is positively charged at acidic and neutral pH values. Enantiomeric separation of cationic substances is best performed at low pH [31,32]. The baseline resolution was obtained in the pH range 2.5–4.2; worse resolution and faster migration times were observed at higher pH. This electrophoretic behaviour of the analytes is probably related to the change of the EOF. EOF at pH > 4.5 became considerable therefore short migration times were obtained. Accordingly, there was not enough time

Fig. 5. The effect of different pH on the resolution (RS ) of rac-RIV, others as in Fig. 3A.

According to Wren and Rowe’s model [33], there exists an optimum concentration for a chiral selector under which an enantiomeric pair can be best resolved. This optimum concentration is dependent on the binding strength between the chiral selector and the solute, with a weakly interacting solute normally requiring a higher concentration of chiral selector. Due to the weak protonation of RIV in the acidic running buffer free form of RIV tended to migrate toward the cathode site. Hence any apparent mobility direction towards the anode site should be attributed solely to the interactions between the RIV and S-␤-CD. Therefore, the migration times are good indicators of the strength of the interactions concerned. 3.3.1. Development approach The effect of six CD derivates (predominantly ␤-CD derivatives) on electrophoretic separations of two enantiomers was investigated. The CDs examined were: HP-␣CD, HP-␤-CD, HP-␥-CD, S-␤-CD, CM-␤-CD and DM-␤CD. In order to determine the impact of CD concentration on resolution, low and high concentrations were evaluated. Based on a literature source [34] and our experience, 5 mM was selected as an appropriate low concentration. Simultaneously, 30 mM was chosen as a high concentration for neutral CDs for screening. For DM-␤-CD, the concentration had to be lowered to 15 mM in order to maintain reasonably short run times. The comparison was also made for 5, 10 and 15 mM S-␤-CD. Minute differences in resolution were found using these different concentrations and therefore only 7 mM S-␤CD was used in further experiments. 3.3.1.1. Enantioseparation of RIV by neutral CD-modified CZE. The chiral separation of RIV was first explored in acidic phosphate buffers and with various kinds of neutral CDs. Quang and Khaledi carried out chiral separation of basic compounds with different types of ␤-CDs. The water solubility of ␤-CD can be increased and its enantioselectivity can be altered by chemically modifying the CD with alkyl substituents [35,36] that also changes the size and flexibility of the CD cavity [37]. The orientation of the bulky and/or charged substituents on the CD cavity can decrease the enantioselectivity by causing steric hidrance and/or coulombic repulsion, preventing guest–host inclusion complex formation [38]. Using the proposed chiral recognition model [26], it is

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conceivable that the steric interaction between the substituent on the N atom of guest molecules and the functional groups at the C-2 positions of ␤-CD generally decrease the stability of inclusion complexes (comparing DM-␤-CD with ␤-CD). In other words, the steric effect increases the differences in stability of complexes formed by S- and R-enantiomers, leading to an increase in enantiomeric resolution. It is important to note that since ␤-CD contains 21 hydroxyl groups, the products obtained from chemical modifications are always a mixture of the derivatized ␤-CD with various degree of substitution [39]. It is known that the partial alkylation of ␤-CD is more likely to occur at the hydroxyl groups at C-2 and C-6 positions. Since DM-␤-CD and HP-␤-C are partially alkylated products of ␤-CD, they may possess similar molecular structure, leading to their similarity in enantioselectivity. It can be expected that HP-␤-CD resembles DM-␤-CD by having some of the C-2 and C-6 hydroxyl groups substituted, and differs with DM-␤-CD by having longer and polar substituents and by having lesser degree of substitution. Consequently, HP-␤-CD is generally more selective for basic compounds with a chiral center located at a distance from the hydrophobic moiety such as some of ␤-blockers. On the other hand, DM-␤-CD is more suitable for compounds with a chiral center close to the aromatic ring such as ephedrine [40]. It is not surprising that the enantiomers of RIV could not be resolved with ␤-CD. It seems that the presence of a longchain substituent at the meta position prevent the aromatic moiety from penetrating deeply into the ␤-CD cavity, leaving the chiral center of the guest molecule beyond the reach of the functional groups on the rim of ␤-CD cavity. Similarly, no chiral separation for RIV was achieved by using any of the neutral-modified ␤-CD. This might also be due to the steric effect at the meta position of the aromatic moiety, which prevented the functional groups of the guest molecules from interacting with the functional groups on the rim of ␤-CD. Therefore, it is suggested that ␤-CD derivatives with longer substituents or other kinds of chiral selectors be explored for separation of the enantiomers of RIV. It should mentioned that ionization of relevant ␤-CD derivatives is suppressed under pH 2.5. 3.3.1.2. Enantioseparation of RIV with anionic CD-modified CZE. The failure of CZE mode to achieve the chiral separation of RIV prompted us to explore the feasibility of including charged chiral selectors in running buffers. The utility of various charged CD derivatives in chiral separations has been reviewed previously [41]. The charged CD derivatives may act not only as “pseudostationary phases” like the micelles in MEKC separation but also as chiral discrimination agents. Therefore the chiral separation of both charged and neutral compounds is allowed. CMCD is characterized by ∼3–3.5 carboxylic groups having pK of 3.5–4 and thus it can be used in two different modes [42]. At pH < 3.5, the carboxylic acid groups become protonated, resulting in an electrically neutral chiral selector. At

pH > 4 the carboxylic acids become ionized and the negatively charged derivative migrate towards the anode. One of the main advantages of this pH dependent charge of CMCD is the possibility to reverse the migration order of enantiomers. At pH 2.5 the RIV–CMCD complexes have cationic character and thus they migrate toward the cathode while no separation of enantiomers is achieved. Among the various charged chiral selectors, S-␤-CD is one of the most frequently utilized. Randomly substituted S-␤-CD is commercially available and is relatively inexpensive. Stalcup and Gahm demonstrated that a large number of pharmaceuticals could be resolved enantiomerically in a S-␤-CD-modified CZE system [43]. At lower pH (2.5) the intermolecular interactions, especially the hydrogen bonding, became more available between the polar functional groups of the CD and the analyte. Secondly, at lower pH the basic amino group of the analyte would be protonated (positively charged), thus the inclusion CD–analyte complex formed would have a better electrophoretic migration within the applied electric field and would enhance effective separation. Note that the presence of the sulfate substituents on the CDs allows ion-exchange or electrostatic interactions in addition to more traditional inclusion complexation usually invoked when considering stereospecific interactions with CDs. These interactions contribute to the improvement of the enantioseparation of the analyte under study. In this work, both RIV and S-␤-CD were fully charged at pH 2.5 unlike the neutral CDs. When charged molecules interact with each other, Coulomb interactions additionally stabilize the host–guest complexes and selectively influence fitting of the RIV into the CD cavity resulting in higher mobility difference of the enantiomers [44]. The best separation of R/S-RIV was observed unambiguously in the presence of polyanionic S-␤-CD. The effect of several S-␤-CD concentrations (5–15 mM) on the resolution of RIV in phosphate buffer (100 mM, pH 2.5) is shown in Fig. 6. The increasing concentration of S-␤-CD results in faster migration velocities

Fig. 6. Dependence of the resolution (RS ) on the concentration of S-␤-CD. See Fig. 3A for CZE conditions.

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of the negatively charged drug–S-␤-CD complex and better peak shape of the enantiomers. It is known that a difference in the apparent mobility between two enantiomers will reach a maximum at a particular chiral selector concentration, which depends on the affinity of the enantiomers for chiral selector [28]. The minimum concentration of S-␤-CD in buffer necessary for sufficient baseline separation of racemate is 6.5 mM. Considering data shown in Fig. 6 it is clear that the optimum concentration range of selector is quite narrow – approximately 11 mM; in other words, the enantiomers of RIV show the greatest difference in mobility at 11–13 mM of S-␤-CD. The most suitable running buffer resulting in chiral selectivity for the enantiomers with propitious separation time was found to be a phosphate buffer containing 7 mM S-␤-CD with the pH adjusted to 2.5 with TRIS. Although higher concentrations of S-␤-CD favoured successfully enantiomeric separation, the concentration should not be increased unduly. The multiple-charge feature of S-␤-CD can result in too high ionic strength of the electrolyte solution. Therefore, the high concentrations of S-␤-CD would bring an increased production of the Joule heating during the electrophoretic process, which would reduce column efficiency and the signal-to-noise ratio for UV detection.

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higher temperature was due to a decrease in the formation constant of the analyte–chiral selector complex during the chiral separation and an increase in the solute diffusion. Apparently the complex-formation interaction of the basic drug with S-␤-CD was exothermic reaction and lower temperature supported formation of the complex. Therefore the capillary temperature was maintained at 18 ± 0.1 ◦ C to optimise the analysis of the racemic drug. 3.6. Effect of capillary length on separation The effect of capillary length on the resolution of R/SRIV was studied using capillaries with the effective length of 20, 40 and 50 cm. The 50 cm capillary allowed the best resolution of the peaks according to expectation. However, the improvement in the resolution was achieved at the expense of a longer migration time. It was found that the migration time obtained with 20 cm capillary was 8 min, with 50 cm capillary about 30 min. That is usual effect of capillary length on the mobility of analyte, e.g., longer capillaries give better resolution because they allow to using higher voltage. Fig. 2 illustrates chiral separations of rac-RIV carried out at 25 kV with 30 cm × 50 ␮m i.d. (A) and 30 kV with 60 cm × 50 ␮m i.d. PAA-coated capillary (B).

3.4. Effect of analytical voltage The electrophoretic velocities are directly proportional to the electric field strength. The effect of four voltage values (15, 20, 25 and 30 kV) on migration times was studied (Fig. 7). The limiting factor here is Joule heating. The results showed that the resolution of enantiomers was decreased with increasing the voltage. The optimal voltage was set at 20 kV, which ensured short migration time, acceptable current generated and good resolution. 3.5. Effect of capillary temperature on separation Changes in the capillary temperature can lead to changes in the mobility of analyte. The reduction of resolution at

3.7. Validation The CZE method was validated in terms of linearity, limit of detection (LOD), limit of quantification (LOQ), precision and accuracy. The linear relationship between the concentration and the corresponding peak area was examined in the range of 5–500 ␮M of individual enantiomer and CZE data were evaluated by linear regression. The linear regression parameters of the calibration line are shown in Table 1 (n = 5); they indicate that good linearity (r = 0.9994–0.9995) between Y (peak area) and X (concentration of the particular enantiomer in mM) is attainable over the range tested. The relative standard deviations (R.S.D.) of the peak area and the migration time of each enantiomer for six replicate injections at concentration level 100 ␮M were 2.06 and 1.40% for S-RIV, respectively 1.72 and 0.78% for R-RIV. The precision of the method was satisfactory as indicated by the calculated R.S.D. values. The detection limit (LOD) was determined as concentration giving three times the signal-to-noise ratio and limit of quantification (LOQ) as concentration causing 10 times Table 1 Regression analysisa for the determination of rac-RIV

Fig. 7. The effect of the applied voltage on the migration time. For the separation conditions, see Fig. 3A.

Analyte

Regression equation a Y = (a ± s)X + (b ± s)

Correlation coefficient (r)

S-RIV R-RIV

Y = (604000 ± 13000)X + (8700 ± 6800) Y = (523000 ± 13000)X + (8500 ± 6700)

0.9995 0.9994

a Y: peak area (AU); X: analyte concentration (mM); a: slope; s(a): standard deviation of the slope; b: intercept; s(b): standard deviation of intercept.

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Table 2 Determination of S-RIV in the commercial preparation (Exelon capsules) including the recovery results Declared content (mg/caps)

Found (CZE) (mg/caps)

R.S.D.a (%)

Added (mg ml−1 )

Found (mg ml−1 )

Recovery (%)

R.S.D.a (%)

1.50

1.48

1.60

0.025, 0.050

0.0256, 0.0481

102.4, 96.2

2.10, 1.80

a

Six injections.

the signal-to-noise ratio. At detection wavelength 200 nm the LOD is 5 ␮M for both enantiomers and LOQ is 13 ␮M. 3.8. Application The developed method was used for determining of S-RIV in formulation Exelon (capsules). A typical electropherogram for the analysis of RIV in the formulation is shown in Fig. 8. Both of enantiomers of RIV were baseline-separated within 8 min using the optimised conditions. The sensitivity of the method is sufficient to assay a limited amount of R-enantiomer as an undesirable impurity (≥0.1%). Fig. 9 shows the separation of R/S-RIV (minor peak 0.1%). The results are summarised in Table 2 including the repeatability data. All analytical values fall within 96.2–102.4% of the RIV labelled value. The accuracy of the method was tested by the technique of the standard addition of reference S-RIV. The recoveries of the added S-RIV (150, 300 ␮M) ranged between 96 and 103%. The results demonstrate that the CZE method is a useful, simple a rapid technique for the assay of S-RIV in pharma-

ceutical. In addition, the proposed method also is applicable to the chiral purity control of Exelon capsules.

4. Conclusion The optimised CZE method could be used successfully for the separation of basic compound, namely RIV with a low-pH phosphate buffer and a low-EOF in a PAA-coated capillary. Several CD-types of chiral selectors were examined in order to achieve enantioseparation of R/S-RIV. It has been demonstrated that charged CDs were much more effective in the enantioseparation compared to native forms. The complete separation of the enantiomers was achieved using commercially available randomly substituted S-␤-CD as chiral selector. Efficient chiral separation of RIV was achieved within 8 min with 7 mM S-␤-CD phosphate buffer of pH 2.5, 20 cm PAA-coated capillary operated at 20 kV (18 ± 0.1 ◦ C) and detection wavelength of 200 nm. The CZE technique described in this study was suitable for the determination of low concentration of R-RIV as chiral impurity in biologically active S-RIV.

Acknowledgements This project is financially supported by the Research project LN00B125 of the Czech Ministry of Education, the project FRVS, Grant No. 967/2004/G-6 and Internal Grant Agency of the Ministry of Health of the Czech Republic, No. NL/7689-3/2003.

References Fig. 8. Electropherogram of S-RIV in the pharmaceutical (Exelon capsules). See Fig. 3A for CZE conditions.

Fig. 9. CZE separation of S-RIV and R-RIV present in the concentration ratio of 1000:1. See Fig. 3A for other CZE conditions.

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