A ligand exchange HPLC method for enantioseparation of ofloxacin enantiomers was developed by a using C8 stationary phase. Chromatographic separation was performed on a Zorbax-300SB-C8 column eluted with the mobile phase consisting of 15 % methanol in water containing 10mM phenylalanin and 5 mM CuSO4 at flow rate of 0.8 mL/min. The method was simple, rapid and suitable for routine analytical studies of ofloxacin enantiomers

Dat, Nguyen Tien

Hong Hanh, Tran Thi

Vuong, Nguyen Quoc

Vietnam Academy of Science and Technology: Journals Online

 65 
VIETNAM JOURNAL OF CHEMISTRY         VOL. 53(2e) 65-68                                               APRIL 2015 
DOI: 10.15625/0866-7144.2015-2e-015 
  
 
 
 ENANTIOSELECTIVE SEPARATION OF OFLOXACIN BY LIGAND 
EXCHANGE CHROMATOGRAPHY 
 
Nguyen Tien Dat
*
, Nguyen Quoc Vuong, Tran Thi Hong Hanh 
Institute of Marine Biochemistry, Vietnam Academy of Science and Technology  
Received 23 January 2015; Accepted for Publication 18 March 2015 
 
Abstract 
A ligand exchange HPLC method for enantioseparation of ofloxacin enantiomers was developed by a using C8 
stationary phase. Chromatographic separation was performed on a Zorbax-300SB-C8 column eluted with the mobile 
phase consisting of 15 % methanol in water containing 10mM phenylalanin and 5 mM CuSO4 at flow rate of                
0.8 mL/min. The method was simple, rapid and suitable for routine analytical studies of ofloxacin enantiomers. 
Keywords. Ofloxacin, levofloxacin, ligand exchange HPLC, enantio selective separation. 
 
 
1. INTRODUCTION 
 
Ofloxacin is one of the most commonly used 
second-generation fluoroquinolones due to its wide 
spectrum of antimicrobial activity, low toxicity, long 
half-life, and high stability. As the compound 
contains a chiral center at C-3, ofloxacin is a 
racemic mixture of (R)-ofloxacin and (S)-ofloxacin 
(levofloxacin). The S-enantiomer shows 8–128 times 
higher activity than the R-form against Gram-
positive and Gram-negative bacteria [1, 2]. 
Therefore, enantiomer selective analysis of 
ofloxacin is important for the drug quality control, 
investigation of the pharmacokinetics of the 
enantiomers in vitro as well as for the design and 
development of new chiral pharmaceutics. Several 
analytical methods for determination of ofloxacin 
enantiomers have been reported including capillary 
electrophoresis (CE) [3, 4], and high performance 
liquid chromatography (HPLC) using chiral 
stationary phases or chiral mobile phase additives 
[5,6]. HPLC analysis using chiral stationary phase 
requires a special chiral column, which is expensive 
and only suitable for several enantiomers in most 
cases. In contrast, HPLC methods based on chiral 
mobile phase additives are not only efficient tools 
for the separation of racemic drugs but also 
relatively cheap and feasible. Most of reports have 
shown that the use of RP18 columns eluted by ligand 
exchanged mobile phases consisting of an amino 
acid such as L-leucine or L-phenylalamine provided 
the best separation [6-8]. In the present paper, we 
described the development of a simple and rapid 
HPLC method for separation of ofloxacin 
enantiomers by using a reverse phase C8 column 
and a mobile phase of methanol-water containing 5 
mM copper sulfate and 10 mM L-phenylalanine. 
 
   
Fig. 1: Structures of levofloxacin and ofloxacin 
 
2. EXPERIMENTAL 
 
2.1. Chemicals and reagents 
 
Racemic ofloxacin và levofloxacin (> 98 %) 
were purchased from Sigma-Aldrich (St. Louis, MO, 
USA). HPLC grade methanol, water, and analytical 
grade copper sulfate and L-phenylalanine were 
purchased from Merck (Darmstadt, Germany). Stock 
standard solutions of ofloxacin và levofloxacin were 
prepared by dissolving 1 mg powder in 1 mL 
mixture of methanol-water (1:1 v/v). 
 
2.2. HPLC experiments 
 
HPLC was performed on an Agilent 1200 liquid 
chromatography system equipped with a diode array 
detector. Data acquisition, processing and instrument 
 66 
VJC, Vol. 53(2e), 2015                                                                                                    Nguyen Tien Dat, et al. 
control were monitored by Microsoft Windows XP 
based ChemStation software. Chromatographic 
separation was carried out in a Zorbax-300SB-C8 
column (4.6x250 mm) with a C8 guard column 
maintained at 21 °C. The elution was performed 
with a 30 min. isocratic mobile phase of 15% 
methanol in water containing 10mM phenylalanin 
and 5mM CuSO4 at flow rate of 0.8 mL/min. The 
injection volume was of 10 µL. The DAD 
acquisition wavelength was set at 290 nm.  
Retention factor (k), enantioseparation factor (α), 
and resolution (R) were calculated using the 
following equations: k = (t−t0)/t0, where t and t0 are 
the retention times of analyte and unretained solutes, 
respectively; α = kR/kS, where kS and kR are the 
retention factors of (S)- and (R)-enantiomer, 
respectively; R = 2(tR−tS)/(wR+wS), where tS and tR 
are retention times of the (S)- and (R)-enantiomer, 
respectively, and wS and wR are the baseline peak 
widths of the two enantiomers. 
 
3. RESULTS AND DISCUSSION 
 
3.1. Reverse phase column selection 
 
Previous ligand exchange HPLC methods have 
used only C18 reverse phase columns for 
enantioseparation of ofloxacin. However, our 
experiment showed that C18 columns such as 
Zorbax Eclipse XDB C18 column, YMC Pack Pro 
C18 and J’sphere ODS-H80 did not provide 
separation of ofloxacin enantiomers by using mobile 
phase of 15 % methanol in water containing 10 mM 
phenylalanin and 5 mM CuSO4. This could be 
explained as the amino acid phenylalanin was not 
sensitive with C18 columns [7]. We next tried the 
Zorbax-300SB-C8 column with the mobile phase 
described above. The retention factor (k), 
enantioseparation factor (α), and resolution (R) 
obtained from test columns were compared. As 
showed in table 1, Zorbax-300SB-C8 column 
provided highest resolution (R = 1.56), while no 
separation was found when using C18 columns. 
 
3.2. Mobile phase optimization 
 
Among organic solvents such as methanol, 
acetonitril, ethanol and isopropanol, methanol 
provided the best separation. The concentration of 
methanol was also investigated. Increased organic 
solvent decreased the separation and low methanol 
concentration caused the extended retention time. 
Hence, it is necessary to adjust the methanol 
concentration in the mobile phase in order to obtain 
good and rapid chromatographic separation of 
levofloxacin and (R)-enantiomer. Considering the 
retention time and enantioselectivity, 15% methanol 
was selected as the organic modifier in this work. 
The amounts of copper sulfate and L-phenylalanine 
were set up as 5 and 10 mM, respectively. The pH of 
the mobile phase is another critical factor for the 
enantioseparation. In order to investigate the effect 
of the pH of mobile phase on the enantioselectivity 
of ofloxacin enantiomers, the pH-dependence of the  
nantioseparation was investigated in a pH range of 
3.0-5.2 using phosphoric acid.  
Table 1: Effect of columns on the separation of ofloxacin enantiomers 
Columns kS kR  R 
Zorbax-300SB-C8 3.63 4.18 1.15 1.56 
Zorbax Eclipse XDB C18 6.92 6.92 1.00 - 
YMC Pack Pro C18 7.32 7.41 1.02 <0.1 
J’sphere ODS-H80 7.55 7.55 1.00 - 
 
Previous study indicated that the resolution of 
ofloxacin enantiomers distinctly decreased from 
2.35 to 1.10 when the pH of the mobile phase was 
lowered from 4.0. On the other hand, when the pH 
of the mobile phase exceeded 5.0, Cu
2+
 would 
precipitate and block the chromatographic system 
[7]. Hence, 4.5 were chosen as the optimized pH of 
the mobile phase. With these conditions, 
levofloxacin and (R)-ofloxacin were well separated 
at the retention time of 16.6 and 18.7 min, 
respectively (figure 2). 
3.3. Method validation 
 
Calibration curves were constructed using the 
areas of the chromatographic peaks measured at 
increasing concentrations in the range of 2–200 
μg/mL for ofloxacin enantiomers. The results 
showed good linearity with the correlation 
coefficients of 0.9996 and 0.9991 for levofloxacin, 
and (R)-ofloxacin, respectively. The intra- and 
interday accuracy and precision of the assay 
assessed as relative standard deviation (RSD) were 
 67 
VJC, Vol. 53(2e), 2015                                                                     Enantioselective separation of ofloxacin… 
determined on the basis of peak-area by assaying the 
standards at three different concentrations in five 
replicates in the same day and consecutive days. The 
results showed that the intraday and interday RSDs 
of the proposed method were lower than 1.47 % and 
2.82 % for levofloxacin, and 1.35 % and 4.01 % for 
(R)-ofloxacin, respectively.  The limit of detection 
(LOD) and limit of quantification (LOQ) were both 
determined as the concentrations that produced 
signal-to-noise ratios of 3 and 10, respectively. The 
value obtained for LOD and LOQ were 1.51 and 
5.14 μg/mL, respectively, for two enantiomers. 
 
 
 
Fig. 2: HPLC chromatograms of levofloxacin (A), ofloxacin racemic (B) and  
mixture of levofloxacin-ofloxacin racemic (C) 
 
4. CONCLUSION 
 
A sensitive, simple, and accurate method for 
enantioseparation of ofloxacin enantiomers was 
developed by chiral ligand exchange RP-HPLC 
using Zorbax-300SB-C8 column. The method 
provided good enantioseparation factor (α = 1.15) 
and resolution (R = 1.56) with the mobile phase of 
15% methanol in water containing 10mM 
phenylalanin and 5mM CuSO4 at flow rate of 0.8 
mL/min. This is the first time that the separation of 
ofloxacin enantiomers by using a C8 stationary phase 
has been reported. 
 
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VJC, Vol. 53(2e), 2015                                                                                                    Nguyen Tien Dat, et al. 
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Corresponding author:  Nguyen Tien Dat 
Institute of Marine Biochemistry, Vietnam Academy of Science and Technology  
18 Hoang Quoc Viet, Cau Giay, Hanoi, Vietnam 
E-mail: ngtiend@imbc.vast.vn. 
 


Enantioselective separation of ofloxacin by ligand exchange chromatography

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Enantioselective separation of ofloxacin by ligand exchange chromatography

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