oup_chrsci_bmy061 835..845 ++

Journal of Chromatographic Science, 2018, Vol. 56, No. 9, 835–845 doi: 10.1093/chromsci/bmy061 Advance Access Publication Date: 21 June 2018 Article

Article

Mohamed Nadjib Rebizi1, Khaled Sekkoum1, Nasser Belboukhari1, Abdelkrim Cheriti2, and Hassan Y. Aboul-Enein3,* 1

Bioactive Molecules & Chiral separation Laboratory, University of Bechar, Bechar 08000, Algeria, Phytochemistry & Organic Synthesis Laboratory, University of Bechar, Bechar 08000, Algeria, and 3 Pharmaceutical and Medicinal Chemistry Department, Pharmaceutical and Drug Industries Research Division, National Research Center, Dokki, Giza 12622, Egypt 2

*

Author to whom correspondence should be addressed. Email: [email protected]

Received 14 November 2017; Revised 1 May 2018; Editorial Decision 28 May 2018

Abstract The enantioseparation of three fluoroquinoline antibacterial drugs, namely, flumequine, ofloxacin and lomefloxacin using high-performance liquid chromatography was optimized on seven polysaccharide-derived chiral stationary phases, namely, Chiralpak® IB, chiralpak® IA, Chiralpak® AD, Chiralcel® OJ, Chiralcel® OD, Chiralcel® OD-H and Chiralcel® OZ-3 and applying different mobile phases in isocratic mode is described. The role of addition of organic additives was also investigated. A baseline separation of flumequine, ofloxacin and lomefloxacin enantiomers was achieved. Parameters influencing enantioseparation including mobile phase, organic additive and chemical nature of the chiral selector found to be highly influencing on the enantiomeric separation were investigated. Chiral recognition mechanism(s) are also presented.

Introduction Chirality has become increasingly important topic issue in drug research and has attracted increasing consideration in the pharmaceutical industry. The implication of the chirality is good recognized as stereoisomers. Drug enantiomers show different behaviors in pharmacological activities, pharmacokinetic processes and toxicity (1–3). And that the desired biological activity of enantiomers is mostly restricted to one of the enantiomers. Frequently, one of the enantiomers will be biologically active (and is called « eutomer ») while, the other (called « distomer ») can exhibit unexpected adverse reactions, antagonistic activities or toxic effects (4, 5). Several regulatory agencies such as US Food and Drug Administration (6, 7), the European Medicines Agency (8) and regulatory authorities in China and Japan have set more stringent guidelines for marketing racemic drugs, indicating that preferably only the active enantiomer (eutomer) of a chiral drug should be brought to the market (4, 9, 10). As a consequence, the determination of stereochemical composition and/or the stereochemical purity

of a compound is an important issue in the pharmaceutical, chemical and cosmetic industries (11). However, the development of methods for the quantitative analysis of chiral compounds and for the assessment of enantiomeric purity is extremely challenging because enantiomers possess the same physical and chemical properties which make discriminating and separating them difficult (12, 13). Among various techniques, the chromatographic techniques had a great impact on the determination of stereochemical purity of compounds. The first analytical scale chromatographic enantioseparation was reported by Gil-Av in 1966 (14). Liquid chromatographic separation of the enantiomers on chiral stationary phases (CSPs) is considered to be one of the most accurate and convenient means inherent to this technique in determining the enantiomeric purity of chiral compounds (15) and has advanced rapidly in the last years with the development of various CSPs. Many racemic pharmaceutical compounds can be efficiently resolved by liquid chromatography (LC) using polysaccharide-based CSPs (4, 16, 17).

© The Author(s) 2018. Published by Oxford University Press. All rights reserved. For Permissions, please email: [email protected]

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Liquid Chromatographic Enantioseparation of Some Fluoroquinoline Drugs Using Several Polysaccharide-Based Chiral Stationary Phases

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Separation of chiral fluoroquinolines was reported in past few years (26, 38). The aims of this study are to develop a fast direct resolution for three racemic fluoroquinolines, namely, flumequine, Lomefloxacin and ofloxacin by HPLC using seven polysaccharide-based CSPs, namely, Chiralcel®OZ-3, Chiralcel® OD, Chiralcel® OJ, Chiralcel® OD-H, Chiralpak® AD, Chiralpak® IA and Chiralpak® IB under isocratic mode using different mobile phases and mobile pahse additives. Furthermore, the chiral recognition mechanisms involved between the analyes and the chiral selectors used in this study are discussed.

Experimental HPLC instrumentation and methods The enantiomeric composition of the drug solution was measured by a high-performance liquid chromatograph Shimadzu LC-20A (Shimadzu, Japan) equipment system including an injector with 20 μl rheodyne 1907 sample loop, a pump LC-20A, a vacuum degasser DGU-20 A5 and a Shimadzu SPD 20A variable-wavelength UV photodiode array detector. The chromatographic and integrated data were recorded with LC solution software. The mobile phase for HPLC was filtered through a Millipore membrane filter (Bedford, MA; 0.5 μm) and degassed before use. Various mobile phase systems were investigated in this study. All of them were composed of commonly used organic HPLC-grade solvents: acetonitrile, ethanol, methanol, isopropanol, dichloromethane, ethanol–hexane mixtures and isopropanol–hexane mixtures. The chromatographic runs were performed at a room temperature of ˜20°C. All analytes were dissolved in methanol at concentrations of 1 mg/ml. Injection volume was 20 μl and UV detector was set at 292 nm for lomefloxacin, 300 nm for ofloxacin and 326 nm for flumequine. Chromatographic separations were carried out under isocratic mode at a flow rate of 0.4 ml/min except in case of Chiralcel® OZ-3, the flow rate was 0.5 ml/min.

Chiral stationary phases Separation of the enantiomers was accomplished using seven polysaccharide-based CSPs, namely, Chiralcel® OZ-3, Chiralpak® AD, Chiralpak® IA, Chiralpak® IB, Chiralcel ® OD-H, Chiralcel ® OD and Chiralcel® OJ, which were obtained from Chiral Technologies Europe (Illkrich Cedex, France). The CSPs Chiralpak® AD, Chiralcel® ODH and Chiralcel® OD are coated on silica and are based on tris (3.5dimethylphenylcarbamate) derivatives of amylose and cellulose, Chiralcel ® OZ-3 is coated on silica and based on cellulose tris (3-chloro-4- methylphenylcarbamate), Chiralpak® IA and Chiralpak® IB are immobilized on silica and are based on tris (3.5-dimethylphenylcarbamate) derivatives of amylose and cellulose, respectively,

Figure 1. Chemical structures of studied fluoroquinoline antibacterials. (A) Ofloxacin, (B) Lomefloxcin and (C) Flumequine.


The research and development of quinolone antibacterial agents have seen an enormous worldwide effort for more than 40 years. Over 10,000 structurally related compounds have been isolated and described in patents and scientific papers (17). Quinolone antibacterial agents, a group of synthetic drugs with bactericidal action, inhibit the bacterial growth by interfering with the bacterial enzyme DNA gyrase needed for its DNA synthesis (18, 19). The quinolones have been described as the, “antimicrobial class of the decade.” Their main therapeutic uses are in treating respiratory and urinary tract infections (20). The first quinolone to be marketed was nalidixic acid which was introduced in 1962. Both nalidixic acid and cinoxacin were classified as the first-generation quinolones and are mainly used for urinary tract infections (21). Furthermore, several more other potent fluorinated quinolones have been developed and among them are ofloxacin, lomefloxacin and flumequine. These compounds in vitro exhibit a broad spectrum of activity against Gram-negative and Gram-positive microorganisms and again have lower minimum inhibitory concentration values (22). Many of the fluoroquinoline compounds are chiral in nature (23). Chirality to these compounds is either imparted by presence of stereogenic center in the core part (e.g., flumequine, ofloxacin, etc.) or side chain part (e.g., lomefloxacin, etc.). Several methods have been reported for enantiomeric separation of these compounds (24, 25). Ofloxacin chemically known as (±)-9-fluoro-2.3-dihydro-3-meth yl-10-(4-methyl-1-piperazinyl)-7-oxo-7H-pyrido[1,2,3-d-e]-1,4-benzoxazine-6-carboxylic acid (Figure 1a) is a potent third-generation broad-spectrum fluoroquinoline antibacterial agent (26). It is one of the most frequently used fluorinated quinolone antibiotics (27). Ofloxacin is administered as a racemic mixture. The stereochemical configuration of ofloxacin affects antibacterial activity where the S-(−)-isomer is 8–128 times more active than that of the R-(+)-isomer (9, 28–30). And approximately two times more active than that of th e racemate (10, 31, 32). Several high-performance liquid chromatography (HPLC) and capillary electrophoresis (CE) have been documented in determination and enantioseparation of ofloxacin (33, 34). Lomefloxacin chemically known as (±)-1-ethyl-6,8-difluoro- 1.4dihydro-7-(3-methyl-1-piperazinyl)-4-oxo-3-quinolinecarboxylic acid (Figure 1b) is a chiral quinolone carboxylic acid antibiotic which is marketed as racemate. The enantiomers of Lomefloxacin possess equipotent antimicrobial activity in vitro. Although the enantiomer potencies of Lomefloxacin appear to be similar, yet the pharmacokinetics of Lomefloxacin may be stereoselective (35). Flumequine is chemically known as (±)-9-Fluoro-6,7-dihydro-5methyl-1-oxo-1H,5H-benzo-[i,j]quinolizine-2-carboxylic acid (Figure 1c) is a synthetic antibacterial drug which may be considered as a hybrid of nalidixic acid and ofloxacin (36, 37) reported that the (S)-enantiomer is responsible for the antimicrobial effect and being more effective than the (R)-isomer.

Rebizi et al.

Liquid Chromatographic Enantioseparation of Some Fluoroquinoline Drugs Chiralcel® OJ is coated on silica and based on cellulose (4-methylbenzoate), while Chiralpak® IB contains the same chiral selector as in Chiralcel® OD-H, but the polysaccharide is immobilized onto silica. The dimension of the columns was 250 × 4.6 mm i.d except Chiralcel® OZ-3 column was 50 × 4.6 mm i.d and the particle size was 3 μm for Chiralcel®OZ-3, 5 μm for Chiralcel® OD-H, Chiralpak® IA and Chiralpak® IB and 10 μm for the other columns. The mobile phases were based on solvents compatible with the coated and immobilized polysaccharide CSPs.

All solvents used in the experiment were HPLC or analytical grade and purchased from Sigma-Aldrich (Seelze, Germany) acetic acid (AcOH) from Reidel de Haën (Seelze, Germany), triethylamine (TEA) and ethylamine were from VWR PROLABO (Fontenay-sousBios cedex, France). Samples of ofloxacin (Sanofi-Aventis,Paris, France), lomefloxacin hydrochloride (Biocodex,Gentilly, France) and flumequine (Gerda Laboratories, Paris, France) were obtained and used as received from their respective manufacturers. Accurately 1.0 mg of drug powdered sample was dissolved in 10 ml of methanol. The solution was filtered through 0.45 -μm microporous membrane. The filtrate was subjected for HPLC analysis. Tables I–III show the results obtained with the various columns. It has not been included cases where the retention times exceeded 60 minutes.

Results Three racemic fluoroquinoline drugs were resolved to their respective enantiomers using seven polysaccharide-based CSPs under isocratic condition. Different mobile phases were employed. The polysaccharide-based CSPs used in this study were Chiralcel® OZ-3, Chiralcel® OD-H, Chiralcel® OD, Chiralcel® OJ, Chiralpak® AD, Chiralpak® IA and Chiralpak® IB. The performance of chiral separation is not only related to the structure of chiral selector but also to the properties of organic solvents in the mobile phase used. Therefore, it is important to investigate the influence of different organic solvents on enantioseparation of fluoroquinolines. The influence of both nature of stationary phase and eluent on such parameter was investigated in this study as shown in Tables I–III. These data show insignificant influence of the different solvents on resolution of each sample. In terms of enantioselectivity, retention time and resolution, one can observe that the separation performance for the three analytes in organic solvents is different, which might be related to the solvent polarity and interactions of different organic solvents with the analytes. The enantioselectivity depends on the differences in the relative stabilities and the affinity to the stationary phase. Chiralcel® OZ-3 and Chiralcel® OD-H were found to be suitable for all of the investigated analytes since their respective enantiomers were baseline separated. The enantiomeric separation of flumequine be achieved with any of the columns used in this study except Chiralpak® IB column, while Chiralcel® OZ-3, Chiralcel® OD-H and Chiralpak® IA columns provided higher resolution, especially Chiralcel® OZ-3. The presence of additives is able to improve peak shape. Separation of flumequine enantiomers in polar mode on Chiralcel® OZ- 3 column using mobile phase such as acetonitrile, methanol and ethanol did enhance the chromatographic efficiency

and resolution between the enantiomers. The resolution factor (Rs) and selectivity factor (α) are summarized in Table I. Baseline separation with good resolution (Rs ≈ 4, α ≈ 1.6) was achieved; the typical retention times of flumequine enantiomers were 10 and 15 min in methanol and ethanol. However, in case of acetonitrile, faster elution time was observed, where the retention times for flumequine enantiomers were eluted at 5.5 and 6.9 min, respectively. Furthermore, good resolution (Rs ≥ 3) was obtained with Chiralcel® OD-H in ethanol, Chiralpak® IA and Chiralcel® OD in acetonitrile and with Chiralpak® AD in methanol. Good resolution of ofloxacin enantiomers in polar solvents was achieved when using Chiralcel® OZ-3 and Chiralpak® IA columns (Rs ≈ 4, α ≈ 1.6); the typical retention times for ofloxacin enantiomers were approximately 10 and 19 min. Baseline separation with good resolution (Rs ≥ 2.5, α ≥ 1.4) in acetonitrile was obtained with Chiralcel® OZ-3 and Chiralpak® IA columns, in methanol and with Chiralcel® OZ-3, Chiralcel® OD-H and Chiralpak® IA columns using ethanol as a mobile phase. Lomefloxacin gave the worst resolutions. The results show incomplete separation and poor resolution of the enantiomers. Lomefloxacin failed to resolve on any column with the exception of Chiralcel® OD-H using acetonitrile as a mobile phase where a baseline separation with Rs = 4.25 and α = 1.91 was achieved, while using Chiralcel® OZ-3, a partial separation was observed using acetonitrile with triethylamine and AcOH as additives. Enantioseparation under normal-phase mode using different concentrations of ethanol and isopropanol as organic modifiers in the mobile phase was investigated in order to study the effects of organic modifier on chiral separation. Only one case of good resolution for ofloxacin enantiomers was obtained using Chiralcel® OZ-3 column and ethanol (40℅) Rs = 4.47 and α = 1.62, while lomefloxacin and flumequine enantiomers were separated but with lower resolution values (Rs ≤ 2.5). Surprising high resolution was obtained for flumequine when using Chiralcel® OD-H column (Rs = 19.82 and α = 6.6) (Figure 2) and Chiralpak® IA column (Rs = 23.50 and α = 4.59) while separation of ofloxacin enantiomers was achieved on Chiralcel® OD-H column (Rs = 22.54 and α = 13.12) when 1% ethylamine was added to hexane–ethanol mixture. No indication of separation for the three analytes under study was achieved on Chiralpak® IB column, even upon the addition of acidic or basic additives. However, after the addition of TEA and AcOH as a mobile phase additives, separation was achieved using different pure polar solvents or hexane–ethanol or hexane–isopropanol. The Rs values were slightly increased. Consideration for the amphoteric character of ofloxacin and lomefloxacin, TEA a common additive, was often added to the mobile phase in which the analytes contained an amino basic function, however, the total content of the additives should remain below 0.5%. When triethylamine was added, peak tailing was reduced and Rs was enhanced, with reduction of the retention time (39).

Discussion A polysaccharide-based CSP contained five chiral centers per unit, and fluoroquinolines studied had only one chiral center. The stereoelectronic interactions between the enantiomers and the CSP generated enantioselectivity, thus causing significant differences in the migration of enantiomers inside the column. Interactions between the hydroxy groups of the stationary phases and the side chain groups of the analytes might be necessary for chiral recognition. The


Reagents and chemicals

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Rebizi et al.

Table I. Chromatographic Data for the Separation of Flumequine by Chiral HPLC Column

Mobile phase

®

Chiralcel OZ

Chiralcel OD-H ®

Chiralcel OJ®

Chiralcel OD®

Chiralcel AD®

Chiralpak IB

®

tr1

tr2

k’1

k’2

α

5.50 10.22 10.94 28.99 17.58 11.23 18.15 14.99 09.03

6.98 15.44 16.18 37.24

0.79 3.08 3.05 10.54 0 0.44 1.45 0 0

1.27 5.17 4.99 13.83

1.61 1.68 1.64 1.31 0 2.97 1.16 0 0

3.94 4.34 4.70 2.06 0 3.60 0.49 0 0

10.57 08.30 12.10 12.52 07.73 08.16 12.23 07.88 11.98 8.61 9.09 7.53 22.27 25.68 20.51

21.71 12.97 12.85 13.38

0.23 0.36 0.30 0.57 0 0 0.66 0 0.39 0 0.39 0 2.27 2.44 1.57

1.53 1.13 0.38 0.68

6.60 3.12 1.27 1.19 0 0 1.25 0 4.59 0 2.59 0 1.10 1.23 0

19.82 3.60 1.15 1.21 0 0 2.03 0 23.50 0 0.66 0 0.34 0.64 0

27.40 08.62

37.48 12.38

7.88 0.26

11.15 0.80

1.41 3.27

0.86 3.27

09.95 22.21 22.08 22.14

0 0 0.32 1.53 1.41 1.75

0.36 1.78 1.61 1.95

0 0 1.125 1.168 1.146 1.11

0 0 0.21 0.50 1.02 1.34

18.49 14.46 09.66 20.16 20.31 20.67 50.581 8.89 8.80 12.56 5.78 8.74 7.57 9.08 8.80 8.37 8.80 8.80 9.01 a

17.99 19.86

13.44 24.13 13.15 23.84 29.94

55.180 10.27 10.70 13.44 9.14 9.82 9.29 10.09 11.69 11.69 11.47

34.713 0.36 0.67 0.86 0.20 0.46 0.76 0 0.377 0.40 0.54 0.44 0

a

a

1.31 1.69

0.83 1.81 1.00 2.50 3.01

42.981 0.57 1.04 0.99 0.90 0.64 1.16 0.578 0.789 1.046 0.88 a

1.108 1.58 1.54 1.15 4.57 1.39 0.76 0 1.53 1.95 1.93 1.99 0

Rs

2.715 1.7 2.77 1.12 4.78 1.31 0.86 0 1.59 1.70 2,08 2,3 0

a

a

12.21 11.70

0 0

0 0

0 0

10.18 8.33 8.68

0 0 0

0 0 0

0 0 0

k’, capacity factor; α, selectivity; Rs, resolution. IAP, isopropanol; ACN, acetonitrile; EtOH, ethanol; Hex, hexane; MeOH, methanol; DCM, dichloromethane. a The compounds eluted over 60 minutes.


Chiralpak IA®

ACN+0.4%TEA+0.05%AcOH EtOH+0.4%TEA+0.05%AcOH MeOH 40%IAP+60%Hex ACN EtOH EtOH+0.4%TEA+0.05%AcOH IAP DCM 40%IAP+60%Hex 30%IAP+70%Hex Hex+1% Ethylamine ACN+0.4%TEA+0.05%AcOH EtOH EtOH+0.4%TEA+0.05%AcOH IAP DCM 60%EtOH+40%Hex+0.4TEA+0.05AcOH 40%IAP+60%Hex Hex+1% Ethylamine ACN EtOH IAP 40%EtOH+60%Hex+TEA+AcOH 50%IAP+50%Hex 40%IAP+60%Hex 10%IAP+90%Hex 10%IAP+90%Hex+0.05TEA+0.001AcOH ACN EtOH EtOH+0.4%TEA+0.05%AcOH IAP 60%EtOH+40%Hex+0.4TEA+0.05AcOH 40%EtOH+60%Hex+TEA+AcOH 40%IAP+60%Hex 40%IAP+60%Hex+0.04TEA+0.01AcOH 10%IAP+90%Hex 10%IAP+90%Hex+0.05TEA+0.001AcOH ACN ACN+0.4%TEA+0.05%AcOH EtOH MeOH IAP DCM 60%EtOH+40%Hex+0.4%TEA+0.05%AcOH 50%IAP+50%Hex 45%IAP+55%Hex 35%IAP+65%Hex 30%IAP+70%Hex 10%IAP+90%Hex Hex+1% Ethylamine EtOH EtOH +0.4%TEA+0.05%AcOH IAP 60%EtOH+40%Hex+0.4%TEA+0.05%AcOH 50%IAP+50%Hex 40%IAP+60%Hex

Flumequine

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Liquid Chromatographic Enantioseparation of Some Fluoroquinoline Drugs Table II. Chromatographic Data for the Separation of Ofloxacin by Chiral HPLC Column

Mobile phase

®

Chiralcel OZ

Chiralcel OD-H®

Chiralcel OJ®

Chiralcel OD®

Chiralcel AD

®

Chiralpak IB®

α

tr1

tr2

k’1

k’2

10.42 13.05 16.23 30.08 18.68 09.97 09.61 15.06 07.83

19.74 19.48 24.83 47.24 31.74 10.37 22.20 17.82

2.71 3.87 4.72 12.48 5.93 0.19 0.55 1.10 0

6.02 6.27 7.75 20.18 10.78 0.24 2.59 1.49

2.22 1.62 1.64 1.62 1.82 1.25 4.69 1.35 0

4.19 2.99 5.78 4.47 2.54 0.99 2.08 1.20 0

0 0.15 1.68 2.53

0 2.01 2.61 3.71

0. 13.12 1.55 1.47

0. 22.54 8.12 5.76

0 0

0 0

8.40 9.11 16.71 33.29

23.76 22.50 44.39

07.719 10.316

a

08.90 08.36 13.63 16.07

15.07 06.30 16.40 07.84 10.03 08.39 08.59 a

a

14.68 17.73

17.78

a

08.91 10.59 08.34 08.73 07.20 08.75 08.72 10.50 a

a

0 0

0 0

a

a

a

a

0 0 1.02 1.10

0 0 1.18 1.31

0 0 1.15 1.20

0 0 0.45 0.27

0

0 0 1.20 0 0 0 0

0 0 0.30 0 0 0 0

a

a

a

0 0 0 0 0 0 0 0

0 0 0 0 0 0 0 0

0 0 0.54 0 0 0 0 a

0.67 0

0 0 0 0 0 0 0 0

14.97 23.78 26.18 08.25 08.31 08.75

Rs

a

0 0 0 0 0 0

a

a

a

0 0 0 0 0 0

0 0 0 0 0 0

IAP, isopropanol; ACN, acetonitrile; EtOH, ethanol; Hex, hexane; MeOH, methanol; DCM, dichloromethane. a The compounds eluted over 60 minutes.

side hydroxy groups of the chiral selector of the stationary phases can act as steric barriers or as hydrogen-bonding donor or acceptor groups. The mobile phase also plays an important role in affecting the steric environment of the chiral grooves of the stationary phase that contributes to enantioselectivity. However, polar solvent was likely to cut down the resolution by taking up chiral centers of the CSP or forming hydrogen bonding with the enantiomers instead of

hydrogen bonding between the enantiomers and stationary phase (40). The mechanism of separation using direct chiral separation methods is based on the interaction of CSP with enantiomer that is analyte to form short-lived, transient diastereomeric complexes through interactive forces. The complexes are formed as a result of hydrogen bonding, dipole–dipole interactions, π–π bonding, electrostatic interactions (Van der Waals forces), inclusion complexation


Chiralpak IA®

ACN+0.4%TEA+0.05%AcOH EtOH+0.4%TEA+0.05%AcOH MeOH 40%EtOH+60%Hex+TEA+AcOH 40%IAP+60%Hex ACN EtOH EtOH+0.4%TEA+0.05%AcOH IAP 40%IAP+60%Hex 30%IAP+70%Hex Hex+1% Ethylamine ACN EtOH EtOH+0.4%TEA+0.05%AcOH IAP DCM 60%EtOH+40%Hex+0.4TEA+0.05AcOH 40%IAP+60%Hex Hex+1% Ethylamine ACN IAP 40%EtOH+60%Hex+TEA+AcOH 50%IAP+50%Hex 40%IAP+60%Hex 10%IAP+90%Hex 10%IAP+90%Hex+0.05TEA+0.05AcOH ACN EtOH EtOH+0.4%TEA+0.05%AcOH IAP 60%EtOH+40%Hex+0.4TEA+0.05AcOH 40%IAP+60%Hex 40%IAP+60%Hex+0.04TEA+0.05AcOH 10%IAP+90%Hex+0.05TEA+0.05AcOH ACN ACN+0.4%TEA+0.05%AcOH EtOH IAP 40%EtOH+60%Hex+0.4%TEA+0.05%AcOH 50%IAP+50%Hex 40%IAP+60%Hex 10%IAP+90%Hex Hex+1% Ethylamine ACN+0.4%TEA+0.05%AcOH EtOH EtOH +0.4%TEA+0.05%AcOH IAP 50%IAP+50%Hex 40%IAP+60%Hex

Ofloxacin

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Rebizi et al.

Table III. Chromatographic data for the separation of Lomefloxacin by Chiral HPLC Column

Mobile phase

®

Chiralcel OZ

Chiralcel OD-H ®

Chiralcel OJ

®

Chiralcel OD®

Chiralcel AD®

Chiralpak IB®

tr1

tr2

K’1

18.83 17.48 10.15 12.73 20.07 32.43 08.39 09.35 19.63 7.806 08.16 20.85 22.49

19.97 19.45 14.96 14.92

21.03 29.28

5.52 5.23 4.97 5.20 0 0 0.37 0.62 0 0 0 1.67 0.34

08.37

0 0.65

07.68 08.07

10.44 18.95

08.18 a

a

0 1.70 0.74

1.07 1.13 1.63 1.21 0 0 1.91 3.69 0 0 0 1.01 2.2

0.04 0.90 2.63 0.20 0 0 4.25 1.09 0 0 0 0.04 1.27

0.71

0 1.10

0 0.04

0

0

a

a

0 0 0 0

0 0 0 0

1.83 0

1.16 0

0 0 1.17

0 0 1.14

0 1.69

0 0.88

0 5.89 0

0 0 0

5.91 5.93 3.05 6.27

0.71 2.28

a

0 0 0 0

21.49

0.825 0

30.25 08.42 07.54

08.59

0 0 5.33

21.49 08.90 10.60

11.86

0 0.21

08.71 18.35 08.71 a

Rs

0 a

09.78 08.39 18.75 24.02

15.60 19.29

α

K’2

1.51

6.21

035

0 0 0 a

25.24 08.91 09.39 08.27 08.30 08.66

a

0 0 0 0 0 0

a

a

a

0 0 0 0 0 0

0 0 0 0 0 0

IAP, isopropanol; ACN, acetonitrile; EtOH, ethanol; Hex, hexane; MeOH, methanol; DCM, dichloromethane. a The compounds eluted over 60 minutes.

(41), and steric effects can also achieve better resolution. Though these stationary phases are cellulose or amylose derivatives which are deposited on a support of porous silica, the carbamate group of CSPs plays an essential role since the chiral recognition with the analyte may interact through intermolecular hydrogen bonds and dipole–dipole interactions. Furthermore, the position of the substituents on the phenyl moiety highly affects the ability of chiral recognition of these CSPs (42).

Finally the best enantioseparation of flumequine is obtained using methanol as a mobile phase where a short retention time is achieved without the need of using additives, as shown in Figure 2. Resolution with good peak shape and reasonable migration time was obtained when using Chiralcel® OZ-3. Typical chromatograms for three samples are shown in Figures 2–4. All of the three fluoroquinolines studied have a carboxylic acid group on the fused tricyclic ring system which imparts a degree of


Chiralpak IA®

ACN ACN+0.4%TEA+0.05%AcOH EtOH+0.4%TEA+0.05%AcOH MeOH 40%EtOH+60%Hex+0.4%TEA+0.05%AcOH 40%IAP+60%Hex ACN EtOH EtOH+0.4%TEA+0.05%AcOH IAP 40%IAP+60%Hex Hex+1% Ethylamine ACN Ethanol IAP DCM 60%EtOH+40%Hex+0.4TEA+0.05AcOH 40%IAP+60%Hex Hex+1% Ethylamine ACN IAP 40%EtOH+60%Hex+0.4%TEA+0.05%AcOH 50%IAP+50%Hex 40%IAP+60%Hex 10%IAP+90%Hex 10%IAP+90%Hex+0.05TEA+0.05AcOH ACN EtOH+0.4%TEA+0.05%AcOH 60%EtOH+40%Hex+0.4TEA+0.05AcOH 40%EtOH+60%Hex+0.4%TEA+0.05%AcOH 40%IAP+60%Hex 40%IAP+60%Hex+0.04TEA+0.01AcOH 10%IAP+90%Hex 10%IAP+90%Hex+0.05TEA+0.001AcOH ACN ACN+0.4%TEA+0.05%AcOH EtOH MeOH IAP 60%EtOH+40%Hex+0.4%TEA+0.05%AcOH 40%IAP+60%Hex Hex+1%Ethylamine ACN+0.4%TEA+0.05%AcOH EtOH EtOH +0.4%TEA+0.05%AcOH IAP 50%IAP+50%Hex 40%IAP+60%Hex

Lomefloxacin

Liquid Chromatographic Enantioseparation of Some Fluoroquinoline Drugs

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Figure 2. Enantioseparation chromatograms of flumequine: (A) on Chiralcel® OZ-3, mobile phase: methanol; (B) on Chiralpak® IA, mobile phase hexane+1% ethylamine; and (C) on Chiralcel® OD-H, mobile phase: hexane+1%ethylamine.

rigidity to the substituent, and ofloxacin and lomefloxacin possess a methyl group at the asymmetric C-3 position in the oxazidine ring (26). Flumequine is acidic in nature since the quinolone ring nitrogen does not have any appreciable basicity in aqueous solution; ofloxacin and lomefloxacin are zwitterions, due to the presence of a basic nitrogen at the quinolone ring. However, ofloxacin and

lomefloxacin have two potential ionizable functional groups namely a basic piperazinyl group and a carboxylic acid group. The presence of an ionizable basic nitrogen containing group at the piperazinyl group will be charged under acidic condition. Any charged compounds will have weaker separation due to their solubility in the mobile phase. But in presence of acidic and basic additives,

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Figure 3. Enantioseparation chromatograms of ofloxacin: (A) on Chiralcel® OZ-3, mobile phase: EtOH+0.4%TEA+0.05%AcOH; (B) on Chiralpak® IA, mobile phase etanol; and (C) on Chiralcel® OD-H, mobile phase: EtOH+0.4%TEA+0.05%AcOH.

considerable enantioseparation is obtained. Potentially, problems may arise with HPLC analysis of samples at or around their pKa values, therefore, slight changes in the pH could dramatically change the elution order or peak shape. The separation of peaks is dependent on the mobile phase composition and the type of the stationary phase used. The fluoroquinoline drug enantiomers studied were best resolved by using Chiralcel®

OZ-3 and Chiralcel® OD-H columns. Additionally, a flumequine was separated in all of columns used in this study except Chiralpak® IB column. All other columns failed to provide enantioseparation for lomefloxacin with the exception of Chiralcel® OZ-3 and Chiralcel® OD-H columns. This failure may be due to the remoteness of the chiral center from the carboxylic acid group (26). Lomefloxacin is substituted at position 7 of the bicyclic nucleus with


Figure 4. Enantioseparation chromatograms of Lomefloxacin: (A) on Chiralcel® OZ-3, mobile phase: CAN+0.4%TEA+0.05AcOH and (B) on Chiralcel® OD-H, mobile phase: ACN.

Typical chromatograms for three analytes were observed with Chiralcel® OZ-3 column in different mobile phases. The coated polysaccharide-based stationary phase in Chiralcel® OZ-3 column has higher selectivity than the other columns, being suitable for the enantioselective separation of fluoroquinolines. Representative chromatograms of the enantiomeric resolution of flumequine are shown in Figure 2 (Rs = 4.70) and ideal peak shape with tailing factor 1.64. A relatively important approach is the use of halogenated polysaccharide short column, because of their ability to perform fast chiral separations with polar mobile phase without additives. Chiralcel® OZ-3 is cellulose tris (3-chloro-4-methylphenylcarbamate)-coated CSP on silica gel. The separation of enantiomers on Chiralcel® OZ-3 was due to the interaction between the analyte and the polar carbamate group on the CSP. The carbamate group on the CSP interacts with the analyte through hydrogen bonding using C = O and NH groups present in the CSP and C = O and OH in the fluoroquinolines. In addition, dipole–dipole interaction occurs between the C = O group on the CSP and C = O group on the fluoroquinolines. Therefore, for rapid and efficient chiral separations, the use of short column such as Chiralcel® OZ-3 appears to give a suitable result with some major advantages: short retention time and less solvent consummation and good resolution. Mobile phase additives are widely used in HPLC to regulate analyte retention behavior (43). Adding a certain amount of an organic mobile phase additives to the mobile phase with all solvents was tested in order to improve the symmetry of the peaks. Both acidic additive, e.g., AcOH for weakly acidic chiral drugs and basic additive such as trimethylamine for weakly basic chiral compounds are added to the mobile phase, thereby reducing the degree of ionization of the analyte, thus causing peak tailing improvement and enhanced the column efficiency. The use of acidic or/and basic mobile phase additive results in the baseline stability, peak shape and separation selectivity, which positively affect the performance of the resolution. A mobile phase containing acetonitrile, 0.04% triethylamine and 0.05% AcOH was capable of baseline resolving the peaks of interest in 5~6 min, as shown in Figure 2 (for flumequine enantiomers). Therefore, in this study, the effects of different isopropanol concentration in the mobile phase on enantioseparation of flumequine

Figure 5. The effects of isopropanol concentrations on the resolution of the flumequine in Chiralpak® AD column.


a heterocyclic ring containing a chiral center, which is far away from the quinolone core (26). In contrast, ofloxacin and flumequine enantiomers have a methyl group on their asymmetric carbons, which are located on the oxazine ring. In HPLC, the retention behavior of an analyte is the result of complex interactions with both the polysaccharides’ stationary phases and mobile phase, which is stabilized by Van der Waals forces, hydrogen bonding and hydrophobic interactions. The spatial differences between enantiomers result in interactive selectivity. The mechanism of chiral recognition in the resolution of fluoroquinoline is, however, not yet clear. Although the stationary phase has an active role in the separation process, we focused on mobile phase optimization, because this is the easiest way of adjusting retention and selectivity.

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– A baseline separation of flumequine, ofloxacin and lomefloxacin enantiomers was achieved on several polysaccharide-based CSPs. – Parameters influencing enantioseparation including mobile phase, organic additive and chemical nature of the chiral selector were investigated and found to be highly influencing on the enantiomeric separation. – Chiral recognition mechanism(s) are also presented.

Conclusion The enantioseparation efficiency of three fluoroquinoline antibiotic drugs, namely, flumequine, ofloxacin and lomefloxacin was investigated. All of enantiomers were successfully separated under isocratic-mode HPLC with UV-detection using polysaccharide-based CSPs. Mobile phase and organic additives were optimized. Baseline separation was achieved within 20 min with good separation factor and Rs. Addition of mobile phase additive to the mobile phase results a high efficiency separation with sharp, symmetrical peaks and baseline resolution in a minimum runtime.

Acknowledgments Financial support of the project by the MESRS ALGERIA is gratefully acknowledged.

Conflict of interest statement The authors declare no conflict of interest.

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