Application of rifampicin as a chiral selector for enantioresolution of basic drugs using capillary electrophoresis

Accepted Manuscript Title: Application of rifampicin as a chiral selector for enantioresolution of basic drugs using capillary electrophoresis Author: Shuchi Dixit Jung Hag Park PII: DOI: Reference:

S0021-9673(16)30646-X http://dx.doi.org/doi:10.1016/j.chroma.2016.05.055 CHROMA 357582

To appear in:

Journal of Chromatography A

Received date: Revised date: Accepted date:

21-3-2016 13-5-2016 15-5-2016

Please cite this article as: Shuchi Dixit, Jung Hag Park, Application of rifampicin as a chiral selector for enantioresolution of basic drugs using capillary electrophoresis, Journal of Chromatography A http://dx.doi.org/10.1016/j.chroma.2016.05.055 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Application of rifampicin as a chiral selector for enantioresolution of basic drugs using capillary electrophoresis

Shuchi Dixit and Jung Hag Park*

Department of Chemistry, Yeungnam University, Gyeongsan 38541, South Korea.

* Corresponding author. Tel.: +82 53 810 2360; Fax: +82 53 810 4613. E-mail: [email protected] (J.H. Park), [email protected] (S. Dixit)

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Highlights  Rifampicin was evaluated as a chiral selector for enantioseparation using CE.  Effects of separation conditions for basic chiral analytes were investigated.  Optimal mobile phase conditions for chiral separations were found. Abstract

Rifampicin, a member of rifamycin sub-class of antibiotics which belongs to the naphthalenic ansamycin class of antibiotics, has a characteristic ansa structure, i.e., a ring structure or chromophore spanned by an aliphatic chain. The present work was designed to evaluate its potential as a chiral selector (CS) as its structure consisting of nine stereogenic centers, an aromatic moiety and several functional groups (i.e., one imine, one amide, one acetoxy residue, two aliphatic hydroxyl and three phenolic hydroxyl groups) was expected to instigate multiple enantioselective interactions, namely, hydrogen bonding and inclusion complexation with chiral analytes, and therefore resulting in efficient enantioseparations. Systematic experiments were performed to investigate the effects of concentration of CS, composition of background electrolyte (BGE) and applied voltage on chiral separation. Enantiomers of propranolol and metoprolol were baseline resolved using a BGE consisting of 20 mM CS and 50/50 (v/v) iso-propanol/phosphate buffer (100 mM, pH 7.0) whereas for enantiomers of sertraline, a BGE consisting of 23 mM CS and 40/60 (v/v) isopropanol/phosphate buffer (100 mM, pH 7.0) resulted in baseline resolutions. Key words:

Antibiotics; Capillary electrophoresis; Chiral selector; Enantioseparation;

Rifampicin.

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1. Introduction

The inherent chiral selectivity of biological systems actuates significant differences in the pharmacodynamic, pharmacokinetic and toxicological activities of the enantiomers of bioactive substances [1]. Therefore, the use of a desired enantiomer of a drug is justified clinically for more selective pharmacokinetic profile. International Conference on Harmonization (ICH) has issued guidelines which demand investigation of stereospecific fate of drugs in the body and, enantiomeric purity determination of chiral drugs before their introduction into the market [2]. Hence the development of analytical methods for enantioresolution of pharmaceuticals, and introduction of more diverse, potentially powerful and cost-effective chiral selectors (CSs) for the purpose remain areas of great interest.

Antibiotics are considered a versatile class of CSs as their structural attributes including several stereogenic centers and a variety of functionalities allow efficient enantioselective interactions with a wide range of analytes [3-6]. Rifamycins, the antibiotics belonging to the class of naphthalenic ansamycins, exert activity against a large variety of organisms, such as bacteria and viruses by specific inhibition of bacterial DNA-dependent RNA polymerase [7]. They have a characteristic ansa structure (i.e., a ring structure or chromophore spanned by an aliphatic chain). Various members differ from one another in the type and location of the substituents on their naphthohydroquinone ring (Fig. 1) [8, 9]. Depending on the nature of the substituents

and

pH

of

solution,

these

macrocyclic

antibiotics

can

be

charged

(positively/negatively) or neutral. Owing to their structural features and functionalities that allow multiple interactions (i.e., electrostatic, hydrogen bonding, π-π interactions, etc.) with analytes having widely different structures, rifamycin B and rifamycin SV were employed as CSs [8, 9].

Capillary electrophoresis (CE) is considered an efficient and versatile technique for development of enantioresolution methods [10-12] and to evaluate enantiodiscrimination ability of novel CSs [13-17] due to its several advantages, such as, high efficiency, rapidity and simple instrumentation. The CS is simply added to the background electrolyte (BGE) which gives a solution-based chiral discrimination and hence offers a greater flexibility in the optimization of separation than the stationary phase-based separation, often with higher resolution and chiral 3

selectivity. Method development is quite simple and cost-effective using CE as the run buffer can be altered quickly and effectively, to screen various separation media, with minimal consumption of reagents and samples. Enantioselective ion-pair CE, where primary ionic interaction is accompanied by additional interactions such as hydrogen bonding, dipole-dipole, charge transfer, hydrophobic and steric interactions, for stereoselective formation of diastereomeric ion-pairs between oppositely charged CS and analyte enantiomers, has been reported as an important tool for chiral analysis [18-20]. Literature reveals that cinchona alkaloids, ketopinic and diisoproylideneketogluconic acids and tartrate-boric acid complexes have been successfully employed as ion-pair CSs [18-20].

Taking into account the literature mentioned above and the references cited therein, the present work was designed to evaluate another member of rifamycin sub-class, rifampicin (RMP, Fig. 1) as a CS using CE. Several attributes were considered for its implication as a potential CS, namely, a) possibility of multiple enantioselective interactions with analytes due to presence of nine stereogenic centers, an aromatic moiety and several functional groups including one imine, one amide, one acetoxy residue, two aliphatic hydroxyl and three phenolic hydroxyl groups in its structure, b) prospect of having strong electrostatic interactions with basic analytes, in neutral or basic medium, for existing as an anion (with pKa values of 1.7 and 7.9 related to 4-hydroxyl group and 3-piperazine nitrogen, respectively [21]) in the said range and c) easy commercial availability and cost effectiveness.

2. Experimental

2.1. Apparatus

An Agilent HP

3D

CE System (Palo Alto, USA) equipped with a diode-array UV detector, a

high voltage (±30 kV) power supply and an external nitrogen pressure (up to 10 bars) was used. Other equipment used were Corning 135 pH meter (Corning, USA), Agilent Cary 5000 UVspectrophotometer and Elgastat UHQ water purification system (Bucks, UK).

2.2. Chemicals and materials 4

The chiral analytes, CS (RMP) and other analytical-grade chemicals were obtained from Sigma-Aldrich (Milwaukee, USA) or TCI (Tokyo, Japan). HPLC-grade methanol (MeOH), acetonitrile (ACN) and iso-propanol (iPrOH) were obtained from J.T. Baker (Phillipsburg, USA). Double distilled water (18.2 M·cm) was used throughout. Separations were performed in unmodified fused silica capillaries of 75 µm I.D. and 35 cm total length (25 cm effective length) (Polymicro Technologies, Phoenix, USA). Initial activation of the capillaries was done as per literature report [14]. All solutions were degassed by sonication under vacuum and then filtered through a 0.45-μm filter prior to use.

3. Results and discussion

The present work describes enantioseparation of basic chiral drugs in BGEs composed of i

PrOH/phosphate buffer, and containing RMP as a CS. Systematic experiments were performed

to study the effects of concentration of CS, composition of BGE and applied voltage on enantioresolution by taking propranolol (PRO) and sertraline (SER) as representative analytes. The electropherograms in Fig. 2 show baseline resolutions of the resolved enantiomers under optimized conditions. The analytes were monitored using indirect spectrophotometric detection in account of UV absorption of RMP (minima at approximately 210, 285 and 370 nm [22]). This type of detection usually results in negative peaks (due to a diminished absorbance from a high background) [9]. The polarity of the recorder was reversed so that positive peaks were obtained.

3.1. Concentration of CS

The effect of concentration of CS was studied using run buffers consisting of various concentrations of RMP [i.e., 17, 20 and 23 mM] and results have been summarized in Table 1. Examination of Table 1 and Fig. 3A indicates that an increase in the concentration of CS caused an increase in the migration times of the analytes. The similar pattern has been reported while using other antibiotics as CSs and can be explained by (i) the slowdown of EOF due to increased viscosity of buffer [4-6] and (ii) an effective reduction in overall cathodic migration of CSanalyte complex with an increase in the concentration of CS. As the RMP and analyte molecules 5

exist as anions and cations, respectively in BGEs having pH 7.0, their electrophoretic migration is anodic and cathodic in direction, respectively. However, an increase in the concentration of CS results in its increased complexation with cationic analytes and in turn, an overall reduction in cathodic migration of CS-analyte complex.

An initial increase in the CS concentration resulted in increased resolution (Fig. 3B) which can be attributed to a greater extent of interaction/complexation between the CS and the analyte. However, a further increase from the optimized CS concentration was found to cause a lowering of resolution which can be attributed to saturated complexation between CS and enantiomers leading to peak broadening. In account of the highest resolution values, CS concentrations of 20 mM and 23 mM were considered as optimized concentrations for propranolol and sertraline, respectively.

3.2. Composition of BGE

In view of literature reports showing successful enantioresolution of a wide range of analytes in BGEs consisting of phosphate buffer and iPrOH while using rifamycin B and rifamycin SV as CSs [8, 9]; BGEs consisting of the same components were employed in the present study. Systematic studies were performed to study the effects of concentration and type of organic modifier, and pH of buffer.

Organic modifiers can influence the viscosity of the BGE, solubility and effective charges of analytes and CS, adsorption of CS on capillary wall, and the EOF [3-6]. Moreover, they can modulate the complexation interactions involved in the enantioseparation mechanism when using antibiotics as CSs [23]. Different ratios i.e., 20/80, 30/70, 40/60, 50/50 and 60/40 (v/v) of i

PrOH/phosphate buffer were investigated to study the effect of concentration of organic

modifier. An increase in the concentration resulted in increased migration times of analytes due to decreased EOF (Fig. 3A). The BGEs consisting of 50/50 and 40/60 (v/v) iPrOH/phosphate buffer (100 mM, pH 7.0) resulted in baseline resolutions of PRO and SER, respectively (Table 1). A higher or lower concentration of organic modifiers deteriorated the separation (Fig. 3B)

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which can be attributed to reorganization of the adsorption layer related to solvation of CS and analytes [4]. Examination of Table 1 indicates that exchange of iPrOH with ACN or MeOH did not provide any improvement in enantioresolution. The longest migration times were observed with i

PrOH followed by MeOH and ACN, this trend can be attributed to the order of viscosity of

solvents (i.e., iPrOH> MeOH> ACN). The higher viscosity of constituting organic modifier causes higher viscosity of BGE and results in decreased EOF causing increased migration times of the analytes. In account of the highest resolution values, iPrOH was found to be the best organic modifier.

The chiral recognition properties of antibiotics originate from enantiodiscriminating interactions, such as, hydrogen bonding, dipole-dipole interactions and inclusion complexation which are mostly facilitated by rather stronger non-enantioselective electrostatic interactions [36]. The pH of the running buffer is thus another important experimental parameter to be controlled as it significantly modulates the charges on both the CSs and the analytes and, in turn, the extent of interactions responsible for chiral discrimination. The effective mobility of propranolol was observed to be 5.63×10-2 cm2V-1s-1 (in the absence of CS) and the same for the first eluting enantiomer of propranolol was observed to be 2.03×10-2 cm2V-1s-1 (in presence of 20 mM concentration of CS) in BGEs composed of 50/50 (v/v) iPrOH/phosphate buffer (100 mM, pH 7.0). The effective reduction in overall cathodic migration in the latter case implicates significant interaction between CS and analyte, and formation of CS-analyte complex. The RMP molecules exist as anions in the eluent composed of iPrOH/phosphate buffer (pH 6.5-7.5, as indicated by the electrophoretic mobilities compiled in Table 1) and therefore hydrogen bonding between hydroxyl groups of RMP molecules and amino groups of basic analytes assisted by strong electrostatic interactions between negatively charged RMP molecules and positively charged basic chiral molecules were considered to be the primary interactions responsible for efficient chiral discrimination of basic compounds. The opposite charges on CS and analytes also facilitate enantioseparation as the difference between migrations of CS-enantiomer complex and free enantiomer increases [4]. Table 1 summarizes the effects of pH of run buffer on

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enantioresolution. The highest resolution values at pH 7.0 indicate the most compatible states for aforesaid interactions between the CS and analytes for chiral discrimination.

3.3. Applied voltage

Applied voltage affects EOF, electrophoretic mobilities of analytes, stability of CS and Joule heating [3-6]. An increase in applied voltage resulted in faster migration due to increased EOF and higher migration velocity of analytes. Resolution values increased upon increase of the voltage from 7 to 10 kV. This is likely due to decreased longitudinal diffusion of the analyte molecules as less time is available for the process with increasing EOF [16,17]. Further increase in the voltage resulted in lowered resolution values (Table 1).

4. Comparison with literature reports

Various members of rifamycin sub-class of antibiotics differ from one another in the type and location of the substituents on the naphthohydroquinone ring (Fig. 1). The change of substituents has profound effects on CS's selectivity and efficiency toward charged compounds. The only difference in the structures of rifamycin B and SV is the presence of oxy-acetic acid group and hydroxyl group, respectively as substituents on naphthohydroquinone ring (at position 4). Since the carboxylic and hydroxyl groups on rifamycin B are ionizable (pKa values 2.8 and 6.7 [9]) it exists as a dibasic acid while rifamycin SV is essentially neutral at basic and neutral pHs [9]. Consequently, rifamycin B and SV were able to enantioresolve basic and acidic chiral analytes, respectively [8,9].

RMP has two structural differences with rifamycin B, first, presence of hydroxyl group as substituent (at position 4) as compared to oxy-acetic acid group in the latter and second, presence of an additional methyl piperazine ring substituent (at position 3). Interestingly, these structural features attribute to strong acidic character of hydroxyl group (pKa 1.7) at position 4 in the former due to more efficient delocalization of electron pair. Consequently, better resolution value and shorter migration time of propranolol (i.e., Rs (t1) values of 1.57 (23.06 min) and 1.30 (27.8 min) using RMP (present case) and rifamycin B [9], respectively) can be attributed to stronger 8

electrostatic and hydrogen bonding interactions between hydroxyl groups of RMP and amino groups of basic analytes; although it should be mentioned that comparison is not unequivocal as experimental conditions were not identical in the studies.

Adsorption of antibiotics on the capillary wall of unmodified fused-silica capillaries is considered a common limitation while using antibiotics as CSs as it may cause peak broadening and less reproducible retention times [3-6]. The extent of adsorption increases with presence of more amino groups and overall more positive charge on antibiotics [24]. In the present work with RMP as a CS, the repeatabilities of migration times of separated enantiomers were found to be less than 2.5% intraday and less than 3.5% interday. The repeatabilities of the enantioresolution values were lower than 3.0% for all studied analytes.

5. Conclusions

The antibiotic, RMP, has been successfully explored for its chiral discriminating abilities for the CE enantioseparation of basic analytes. A CS concentration of 20 mM and BGE consisting of 50/50 (v/v) of iPrOH/phosphate buffer (100 mM, pH 7.0) was found to give the best resolution for enantiomers of propranolol and metoprolol; while 23 mM of CS in BGE composed of 40/60 (v/v) of iPrOH/phosphate buffer (100 mM, pH 7.0) was found to be the best for sertraline. The present study indicates that the other members of rifamycin family having different substituents on naphthohydroquinone ring can also be examined for their enantiodiscriminating abilities for multiple-ring containing analytes.

Acknowledgement

This research was supported by Basic Science Research Program through the National Research Foundation of Korea (No. NRF-2015R1D1A1A01057665).

References

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[1] K. M. Rentsch, The importance of stereoselective determination of drugs in the clinical laboratory, J. Biochem. Biophys. Methods 54 (2002) 1-9. [2] ICH‐Topic Q2A: Validation of analytical procedures. International conference on harmonization of technical requirement for registration of pharmaceuticals for human use: Geneva, (1996). [3] S. Dixit, J. H. Park, Application of antibiotics as chiral selectors for capillary electrophoretic enantioseparation of pharmaceuticals: a review, Biomed. Chromatogr. 28 (2014) 10-26. [4] A. F. Prokhorova, E. N. Shapovalova, O. A. Shpigun, Chiral analysis of pharmaceuticals by capillary electrophoresis using antibiotics as chiral selectors, J. Pharma. Biomed. Anal. 53 (2010) 1170-1179. [5] T. J. Ward, A. B. Farris, Chiral separations using the macrocyclic antibiotics: a review, J. Chromatogr. A 906 (2001) 73-89. [6] C. Desiderio, S. Fanali, Chiral analysis by capillary electrophoresis using antibiotics as chiral selector, J. Chromatogr. A 807 (1998) 37-56. [7] H.G. Floss, T. W. Yu, Rifamycin mode of action, resistance and biosynthesis, Chem. Rev. 105 (2005) 621-632. [8] D. W. Armstrong, K. L. Rundlett, G. L. Reid, Use of a macrocyclic antibiotic, rifamycin B, and indirect detection for the resolution of racemic amino alcohols by CE, Anal. Chem. 66 (1994) 1690-1695. [9] T. J. Ward, C. Dann, A. Blaylock, Enantiomeric resolution using the macrocyclic antibiotics rifamycin B and rifamycin SV as chiral selectors in capillary electrophoresis, J. Chromatogr. A 715 (1995) 337-344. [10] P. Jac, G. K. Scriba, Recent advances in electrodriven enantioseparations, J. Sep. Sci. 36 (2013) 52-74. [11] D. A. Tsioupi, S. R. Stefan-van, C. P. Kapnissi-Christodoulou, Chiral selectors in CE: Recent developments and applications, Electrophoresis 34 (2013) 178-204. [12] L. Sanchez-Hernandez, M. Castro-Puyana, M. L. Marina, A. L. Crego, Recent approaches in sensitive enantioseparations by CE, Electrophoresis 33 (2012) 228-242. [13] S. Dixit, J. H. Park, Penicillin G as a novel chiral selector in capillary electrophoresis, J. Chromatogr. A 1326 (2014) 131-138.

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[14] A. P. Kumar, J. H. Park, Azithromycin as a new chiral selector in capillary electrophoresis, J. Chromatogr. A 1218 (2011) 1314-1317. [15] T. Yu, Y. Du, B. Chen, Evaluation of clarithromycin lactobionate as a novel chiral selector for enantiomeric separation of basic drugs in capillary electrophoresis, Electrophoresis 32 (2011) 1898-1905. [16] B. Chen, Y. Du, Evaluation of the enantioseparation capability of the novel chiral selector clindamycin phosphate towards basic drugs by micellar electrokinetic chromatography, J. Chromatogr. A 1217 (2010) 1806-1812. [17] B. Chen, Y. Du, P. Li, Investigation of enantiomeric separation of basic drugs by capillary electrophoresis using clindamycin phosphate as a novel chiral selector, Electrophoresis 30 (2009) 2747-2754. [18] V. Piette, M. Fillet, W. Lindner, J. Crommen, Non-aqueous capillary electrophoretic enantioseparation of N-derivatized amino acids using cinchona alkaloids and derivatives as chiral counter-ions, J. Chromatogr. A 875 (2000) 353. [19] L.J. Wang, J. Yang, G.L. Yang, X.G. Chen, In situ synthesis of twelve dialkyltartrate–boric acid complexes and two polyols–boric acid complexes and their applications as chiral ionpair selectors in non-aqueous capillary electrophoresis, J. Chromatogr. A 1248 (2012) 182. [20] Y. Hedeland, J. Lehtinen, C. Pettersson, Ketopinic acid and diisoproylideneketogulonic acid as chiral ion-pair selectors in capillary electrophoresis: Enantiomeric impurity analysis of Stimolol and 1R,2S-ephedrine, J. Chromatogr. A 1141 (2012) 287. [21] S. Rajabnezhad, L. Casettari, J. K.W. Lam, A. Nomani, M. R. Torkamani, G. F. Palmieri, M. R. Rajabnejad, M. A. Darbandi, Pulmonary delivery of rifampicin microspheres using lower generation polyamidoamine dendrimers as a carrier, Powder Technol. 291 (2016) 366-374. [22] M. A. Lomillo, O. D. Renedo, M. J. A. Martìnez, Resolution of binary mixtures of rifamycin SV and rifampicin by UV/VIS spectroscopy and partial least-squares method (PLS), Chem. Biodiver. 1 (2004) 1336-1343. [23] K. L. Rundlett, M. P. Gasper, E. Y. Zhou, D. W. Armstrong, Capillary electrophoretic enantiomeric separations using the glycopeptide antibiotic, teicoplanin, Chirality 8 (1996) 88-107.

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[24] M. P. Gasper, A. Berthod, U. B. Nair, D. W. Armstrong, Comparison and modeling study of vancomycin, ristocetin a, and teicoplanin for CE enantioseparations, Anal. Chem. 68 (1996) 2501-2514.

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List of Figures Fig. 1. Structures of rifamycin B, rifamycin SV and rifampicin. Fig. 2. Electropherograms showing baseline resolutions of basic chiral analytes using rifampicin as a CS. Conditions: capillary, 35 cm (25 cm effective length) × 75 µm I.D.; BGE, 50/50 (v/v) iPrOH/phosphate buffer (100 mM, pH 7.0) containing 20 mM RMP (for propranolol and metoprolol) and, 40/60 (v/v) iPrOH /phosphate buffer (100 mM, pH 7.0) containing 23 mM RMP (for sertraline); applied voltage, 10 kV; injection, 10 kV, 5 s; capillary temperature, 23ºC; detection, 254 nm. Fig. 3. Effect of concentrations of chiral selector and organic modifier on effective mobility of first eluting enantiomer (μeff1) [A] and resolution (Rs) [B] of propranolol. Conditions: capillary, 35 cm (25 cm effective length) × 75 µm I.D.; BGE, various ratios of iPrOH /phosphate buffer (100 mM, pH 7.0) containing RMP; applied voltage, 10 kV; injection, 10 kV, 5 s; capillary temperature, 23ºC.

13

Fig. 1

14

Fig. 2

15

Fig. 3

16

Table 1. Effect of experimental conditions on separation parameters of enantiomers of basic drugs using rifampicin as a chiral selector Organic modifier

μeo

/buffer (v/v)

μeff1/ μeff2

α

Rs

μeo

μeff1/ μeff2

α

Rs

μeo

μeff1/ μeff2

α

Rs

CS concentration (mM)a 17

PR O

SE R

20

23

20/80

7.4 2

4.79/4.2 4

1.1 6

0.8 6

5.4 5

3.37/3.10

1.0 9

1.1 3

5.0 5

3.31/2.99

1.1 1

1.1 4

30/70

6.6 8

3.86/3.4 5

1.1 2

1.0 1

4.9 9

2.75/2.48

1.1 1

1.2 6

4.6 9

2.65/2.13

1.2 5

1.3 1

40/60

6.3 5

3.60/3.0 3

1.1 9

1.2 1

4.8 1

2.27/1.81

1.2 6

1.3 3

4.0 3

2.18/1.99

1.1 3

1.3 6

50/50

5.9 5

3.17/2.9 4

1.0 8

1.0 3

4.3 0

2.04/1.45

1.4 1

1.5 7

3.8 0

1.90/1.73

1.1 0

1.4 0

60/40

5.8 5

2.98/2.6 1

1.2 6

0.8 6

3.8 8

1.56/1.42

1.1 0

1.2 9

3.6 0

1.27/1.16

1.0 9

0.8 4

20/80

7.4 2

NM

5.4 5

1.33/1.03

1.3 0

1.1 5

5.0 5

0.67/0.51

1.3 0

1.2 1

30/70

6.6 8

NM

4.9 9

1.19/0.79

1.2 1

1.2 2

4.6 9

0.61/0.37

1.7 8

1.3 9

40/60

6.3 5

NM

4.8 1

1.01/0.40

2.5 5

1.4 8

4.0 3

0.56/0.13

4.3 7

2.0 3

50/50

5.9 5

NM

4.3 0

0.91/0.27

3.3 0

1.4 6

3.8 0

0.48/0.19

2.5 0

1.4 7

60/40

5.8 5

NM

3.8 8

0.89/0.46

1.9 3

1.4 3

3.6 0

0.41/0.16

3.1 2

1.4 5

1.4

1.3

pHb 6.5c1 PR

50/50

4.4

2.69/2.1

7.0c2 1.2

1.3

4.3

17

2.04/1.45

7.5c3 1.4

1.5

4.2

1.34/0.91

O SE R

40/60

8

7

4

2

0

4.1 1

0.81/0.3 2

2.7 2

1.4 7

4.0 3

0.56/0.13

1

7

6

4.3 7

2.0 3

3.4 7

0.63/0.21

6

7

4.3 9

1.5 1

Organic modifierd i

PrOH

MeOH

ACN

PR O

50/50

4.3 0

2.04/1.4 5

1.4 1

1.5 7

6.6 4

2.20/1.84

1.2 0

1.2 8

8.7 4

3.48/2.93

1.2 3

0.9 2

SE R

40/60

4.0 3

0.56/0.1 3

4.3 7

2.0 3

6.9 1

1.06/0.85

1.2 6

1.1 3

7.7 4

1.36/1.17

1.1 6

1.0 2

Applied voltagee 7

10

13

PR O

50/50

3.7 0

1.89/1.6 6

1.1 4

1.2 3

4.3 0

2.04/1.45

1.4 1

1.5 7

5.9 4

2.58/2.09

1.2 4

1.4 1

SE R

40/60

3.9 0

0.54/0.1 3

4.1 8

1.4 1

4.0 3

0.56/0.13

4.3 7

2.0 3

5.9 3

0.67/0.54

1.2 4

1.2 2

* PRO, propranolol; SER, sertraline; NM, not measured; Separation factor, α = µeff1/µeff2, where µeff =µapp−µeo = Ltot ·Leff/Vt – Ltot·Leff/Vto (where µeff, µapp and µeo, effective, apparent and electroosmotic mobilities (×10-2cm2V-1s-1) , respectively; Ltot and Leff, total and effective length of capillary, respectively; V, applied voltage; t, t1 and t2 migration times of EOF marker, first and second eluting enantiomers, respectively); Resolution values, Rs = 2(t2–t1)/(w1+w2) (where w1 and w2 are the peak widths at the base of the first and second eluting enantiomers, respectively); Separation conditions: capillary, 35 cm (25 cm effective length) × 75 µm I.D.; capillary temperature, 23ºC; (a) BGE, iPrOH/phosphate buffer (100 mM, pH 7.0) consisting CS; applied voltage, 10 kV; injection, 10 kV, 5 s; (b) BGE, iPrOH/phosphate buffer (100 mM) consisting 20 mM CS (for PRO) and 23 mM CS (for SER); applied voltage, 10 kV; injection, 10 kV, 5 s; (c) Electrophoretic mobilities of CS were -2.60c1, -2.97 c2 and -3.31c3 (×10-2cm2V-1s-1) in BGE consisting of 50/50 iPrOH/phosphate buffer (100 mM), applied voltage, 10 kV; injection, 10 kV, 5 s; (d) BGE, organic modifier/phosphate buffer (100 mM, pH 7.0) consisting of 20 mM CS (for PRO) and 23 mM CS (for SER); applied voltage, 10 kV; injection, 10 kV, 5 s; (e) BGE, iPrOH/phosphate buffer (100 mM, pH 7.0) consisting of 20 mM CS (for PRO) and 23 mM CS (for SER); injection, 5 s.

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