Chromatographia
DOI 10.1007/s10337-014-2699-4
Original
Multiple Response Optimization of a HPLC Method
for the Determination of Enantiomeric Purity of S‑Ofloxacin
Valliappan Kannappan · Sai Sandeep Mannemala 
Received: 28 February 2014 / Revised: 21 April 2014 / Accepted: 18 May 2014
© Springer-Verlag Berlin Heidelberg 2014
Abstract  The aim of the study was to develop a new
HPLC method for direct chiral separation of Ofloxacin
enantiomers using polar non-aqueous mobile phase by
application of response surface methodology. Rotatable
central composite design (CCD) with eight factorial points,
six axial points and six replications in central point was
used to evaluate the influence of three independent vari-
ables (concentration of methanol, diethylamine and flow
rate) on the output responses (capacity factor of first peak,
tailing factors of both the enantiomers, resolution between
the Ofloxacin enantiomers, retention time of the last peak
and chromatographic optimization function). Further, CCD
data were combined with multiple response optimiza-
tion in order to obtain a set of optimal experimental con-
ditions (% methanol/hexane/acetonitrile-43.33/10/46.62
(v/v), % acetic acid/diethylamine-0.4/0.2 and flow rate as
1.4 mL min−1) leading to the most desirable compromise
between resolution and analysis time. The method demon-
strated good correlation between observed and predicted
responses. The developed method was validated according
to ICH guidelines and applied for quantitative analysis of
two commercially available tablets Zenoflox (Ofloxacin)
and Glevo (Levofloxacin). Good agreement was found
between the assay results and the label claim of the mar-
keted formulations by showing good %recovery and %CV.
The study resulted in a better chromatographic system for
the determination of Ofloxacin enantiomers.
V. Kannappan (*) · S. S. Mannemala  age form and biological matrices was performed by capillary
Department of Pharmacy, Faculty of Engineering
and Technology, Annamalai University, Annamalai Nagar,
Chidambaram 608002, Tamil Nadu, India
e-mail: vallikonnect@gmail.com
Keywords  Column liquid chromatography · Ofloxacin
enantiomers · Chiral HPLC separation · Central composite
design · Polysaccharide chiral stationary phase ·
Chromatographic optimization function
Introduction
Chiral drugs constitute one-third of the worldwide marketed
drugs. It is now well documented that the enantiomers of
chiral drugs exhibit different pharmacological and toxico-
logical properties [1]. Therefore, the determination of enan-
tiomeric purity of chiral drugs is important from the point of
view of safety and efficacy of drug therapy [2]. Ofloxacin
(Fig.  1) designated as [(±)-9-fluoro-2,3-dihydro-3-methyl-
10-(4-methyl-1-piperazinyl)-7-oxo-7H-pyridol(1,2,3-de)-
1,4-benzoxazine-6-carboxylic acid] is a second-generation
broad spectrum antibiotic. Ofloxacin possesses one stereo-
genic center in the oxazidine ring and exists in two enan-
tiomeric forms. Levofloxacin (S-OFL) is the S-enantiomer
of Ofloxacin, the eutomer, which shows 8-fold to 128-fold
higher activity than its R-enantiomer (R-OFL) with differ-
ent in vitro bacterial strains [3]. Levofloxacin products may
contain traces of the R-OFL, the distomer, residual from
Levofloxacin synthesis. Hence, it is necessary to identify and
determine both the Ofloxacin enantiomers for chiral quality
control. The monograph of n is official in Indian Pharmaco-
poeia [4], British Pharmacopoeia [5] and USP NF 32 [6]. It
is available in various dosage forms, viz. tablets, eye drops,
infusion, suspension and capsules in Indian market.
The determination of n enantiomers in pharmaceutical dos-
electrokinetic chromatography [7], capillary electrophoresis
[8], ligand exchange methods [9–11]. Also a semiprepara-
tive method by employing immobilized β-cyclodextrin chiral
13

Fig. 1  Structure of n enantiom-
ers
stationary phase (CSP) is reported [12]. However, an inten-
sive literature survey revealed that there are three direct chi-
ral analysis HPLC methods reported for determination of n
enantiomers. Lehr et al. [13] and Rabba et al. [14] developed
methods based on bovine serum albumin (BSA) CSP which
suffers from limitation of usage of delicate column. Sun et al.
[15] proposed a method based on Chiralcel OD-H column,
which has major weakness like excess analysis time (20 min),
low sensitivity (LOD = 10 µg/L) and a poor resolution
(Rs  = 1.3) between the enantiomers. Further, none of these
methods utilized a systematic optimization approach (viz.
chemometric methods) for the separation and quantitation of
the n enantiomers, but employed a time-consuming trial and
error approach which will result only in an apparent optimum
and information regarding the sensitivity of the factors on the
analytes separation and interaction between the factors is not
available. All the above indicated direct chiral HPLC meth-
ods suffer from one or the other limitation and may not be
suitable for routine quality control purpose. Hence, this study
attempts to develop an improved HPLC method for the deter-
mination of Ofloxacin enantiomers using chemometric proce-
dure and also demonstrate the utility of the developed method
by analyzing two commercially available tablets Zenoflox
(Ofloxacin) and Glevo (:Levofloxacin).
Experimental
Apparatus
The chromatographic system from Shimadzu (Shimadzu Cor-
poration, Kyoto, Japan) was used for the analysis which con-
sisted of two LC 20 AD solvent delivery modules, a SPD-M
20A PDA detector and a rheodyne injector (model 7125,
USA) valve fitted with a 20-µL loop. The system was con-
trolled through a system controller (SCL-10A) and a personal
computer using a Shimadzu chromatographic software (LC
Solution, Release 1.11SP1) installed on it. The mobile phase
was degassed using Branson sonicator (Branson Ultrasonics,
USA). A Lux Cellulose-4 column (250 × 4.6 mm, ID) packed
with cellulose tris (4-chloro-3-methylphenylcarbamate)
13
V. Kannappan, S. S. Mannemala
coated on silica particles (5 µm) from Phenomenex (Phe-
nomenex, USA) was used. Absorbance spectra were recorded
using an UV–Visible spectrophotometer (Model UV-1601PC;
Japan) using quartz cell of 1.00 cm of path length.
Software
Experimental design, data analysis and desirability func-
tion calculations were performed by using JMP®, 9.0 trial
version (SAS Institute Inc., Cary, NC) [16]. Perturbation
plot was generated using Design expert®, 8.0 version (Stat-
Ease, MN, USA). The rest of the calculations for the anal-
ysis were performed by the use of Microsoft Excel 2010
software (Microsoft, USA).
Chemicals and Reagents
Working standards of Ofloxacin (±)-Ofloxacin (98.9 %)
was donated by East India Pharmaceuticals (Kolkata, India),
Levofloxacin (99.7 %) was a gift sample from M/S Phar-
maceuticals (Puducherry, India) and probenecid, internal
standard (IS) (99.8 %), was purchased from Sigma-Aldrich
(Bangalore, India); acetonitrile (MeCN), methanol (MeOH)
of HPLC grade and acetic acid (AcOH) and diethyl amine
(DEA) of analytical reagent grade were procured from M/S
SD Fine Chemicals (Mumbai, India). The HPLC grade water
was prepared by using Milli-Q Academic, Millipore (Banga-
lore, India). The tablets Zenoflox (Ofloxacin, 200 mg) from
Mankind Pharma Ltd (Mumbai, India) and Glevo (Levoflox-
acin 500 mg) from Glenmark Pharmaceuticals Ltd (Chennai,
India) was procured from local pharmacy.
Preparation of Solutions and Chromatographic Conditions
Stock and Working Standard Solutions
Stock standard solutions of (±)-Ofloxacin, S-OFL and
probenecid (IS) 1,000 (µg mL−1) were prepared in mobile
phase. The prepared stock solutions were stored at 4 °C pro-
tected from light. Working standard solutions were freshly
prepared by diluting the stock standard solutions with mobile
Multiple Response Optimization of a HPLC Method
phase during the day of analysis. Calibration curves report-
ing peak area ratios of S-OFL and R-OFL to that of the IS
versus drug concentrations were established in the range
of 2–10 µg mL−1 for all the analytes in the presence of
probenecid (10 µg mL−1) as IS. The standard solution pre-
pared for the optimization procedure comprised Ofloxacin
(10 µg mL−1), S-OFL (10 µg mL−1) and IS (10 µg mL−1).
Preparation of Sample Solution
Twenty tablets were weighed and finely powdered. An
amount of tablet powder equivalent to 20 mg of (±)-Ofloxa-
cin was accurately weighed and transferred in a 25-mL vol-
umetric flask; a suitable quantity of IS (25 mg) was added
followed by 12 mL of mobile phase. This mixture was sub-
jected to sonication for 10 min for complete extraction of
drugs and the solution was made up to the mark with mobile
phase and further dilutions were made to obtain a concen-
tration of (±)-Ofloxacin and IS as 8.0 and 10.0 µg mL−1,
respectively. The solution was centrifuged at 4,000 rpm
for 10 min; the clear supernatant was collected and filtered
through a 0.2-µm membrane filter (Gelman Science, India),
and 20 µL of this solution was injected for HPLC analysis.
Chromatographic Conditions
The chromatographic separation was carried out using a
mobile phase consisting of a mixture of MeOH, hexane,
MeCN and small amounts of AcOH and DEA. The analytes
were detected at 285 nm based on isobestic point. Prior to use,
the mobile phase was degassed for 15 min in an ultrasonic
bath and vacuum filtered through 0.45-µm membrane filter
(Gelman Science, India). The injection volume was 20 µL.
The mobile phase was prepared by first mixing the appropri-
ate proportions of MeOH in hexane and then with ACN; sub-
sequently small amounts of AcOH and DEA were added to
this mixture as per design. The HPLC system was used in an
air-conditioned laboratory atmosphere (25 ± 2 °C).
Method Validation
The optimized method was validated according to ICH
Q2(R1) guidelines [17]. The validation parameters like
linearity, limit of detection and quantification, specificity,
accuracy, precision and robustness were addressed.
Results and Discussion
Optimization of Chromatographic Conditions
Rotatable central composite design (RCCD) was employed
in this study for the optimization of chromatographic
separation and to understand the interaction of selected fac-
tors on separation attributes. The selection of key factors
for optimization was based on preliminary experiments and
prior knowledge from literature [18–21]. For instance, the
concentration of hexane was fixed at 10 % v/v and the con-
centration of AcOH was fixed at 0.4 % v/v in the mobile
phase as it aided in maintaining a better peak shape for
both enantiomers. So, the factors selected for optimization
process were % MeOH concentration (A), % DEA concen-
tration (B) and flow rate (C). The factor space of this design
was expanded with in the following range: MeOH concen-
tration was varied from 40 to 60 % v/v, DEA concentration
from 0.2 to 0.3 % and flow rate from 1.0 to 1.4 mL min−1
by keeping concentration of hexane (10 % v/v), AcOH
(0.4 % v/v) constant and % MeCN constituting the rest of
the volume of mobile phase. In order to judge the quality
of the method under different conditions, the following
responses of interest were defined (1) capacity factor of
the first eluted peak (k1), (2) tailing factor of the 2nd peak,
S-OFL (tF2), (3) tailing factor of the 3rd peak, R-OFL
(tF3), (4) resolution between the Ofloxacin enantiomers
(Rs23), (5) retention time of the last peak, R-OFL (tR3) and
(6) chromatographic optimization function (COF).
K
 Rsi
COF = Ai ln + B(tM − tL ) (1)
Rsid
i=1
COF is calculated according to Eq. (1) [22]. Where
Ai and B are weighted parameters (equal to unity in this
study), Rsi is the resolution of the ith pair, Rsid is the
desired resolution for the specific pair (equals to 4), tM rep-
resents the desired maximum analysis time (here assumed
10 min), and tL is the actual time of the last eluted peak. In
the present study, COF was used as it enabled to decrease
data from each chromatogram to a single number which can
be used in the optimization procedure. The elution order
of analytes was confirmed by injecting the pure standards
individually. Probenecid (IS) was used as an internal stand-
ard as it presented acceptable resolution and retention time
with both of the enantiomers.
Table  1 summarizes the conducted experiments,
viz. (n  = 14 + 6) six replicates at center point and the
responses. All experiments were conducted in randomized
order to minimize the effects of uncontrolled variables that
may introduce a bias on the measurements. Two replicates
were performed for each experiment in order to know the
experimental error variance and to test the predictive valid-
ity of the model.
A cross-effect between the three factors (MeOH, DEA
and flow rate) and the selected responses were then ana-
lyzed using a “standard least squares” model. Calculated
coefficients of the response model and obtained P values
are given in Table 2. The insignificant terms (P > 0.05)
13
 V. Kannappan, S. S. Mannemala

Table 1  Central composite Design points Coded factor levels Responses

rotatable design arrangement
and responses %MeOH %DEA Flow rate k1 Rs23 tF2 tF3 tR3 COF

1 0 0 0 0.40 4.7 2.1 1.8 6.4 7.89

2 0 0 0 0.40 4.7 2.1 1.9 6.4 7.89
3 0 0 0 0.41 4.8 2.0 1.7 6.5 7.71
4 0 0 0 0.43 4.7 1.9 1.8 6.5 7.69
5 0 0 0 0.41 4.7 1.9 1.7 6.5 7.69
6 0 0 0 0.40 4.7 1.9 1.8 6.5 7.70
7 0 0 −1.682 0.42 5.2 2.3 1.9 9.2 2.45
8 0 0 +1.682 0.45 4.2 1.6 1.5 5 10.38
9 0 −1.682 0 0.61 4.3 1.9 1.8 6.3 7.47
10 −1.682 0 0 0.57 7.5 1.8 1.5 6.9 7.53
11 +1.682 0 0 0.52 3.6 2.1 1.9 6.8 6.83
12 0 +1.682 0 0.51 4.9 2.1 1.7 6.6 7.68
13 +1 +1 −1 0.45 4.1 2.6 2.1 7.9 4.92
14 +1 −1 −1 0.60 4.1 2.4 2.1 7.6 4.99
15 −1 −1 −1 0.52 5.9 1.9 1.68 8.2 4.39
16 −1 +1 −1 0.43 5.6 2.0 1.6 8.7 3.41
17 +1 +1 +1 0.39 3.9 2.0 1.8 5.7 9.20
18 +1 −1 +1 0.55 3.8 2.0 1.8 5.6 9.14
Central composite rotatable 19 −1 +1 +1 0.48 5.8 1.8 1.5 6 8.97
design arrangements are in 20 −1 −1 +1 0.69 5.7 1.6 1.5 6.9 6.87
random

Table 2  Reduced response models and statistical parameters obtained from ANOVA (after backward elimination)
Responses Reduced response modelsa Adjusted R2 Model P value %CV Adequate precision

k1 k1 = +0.42 − 0.015 A − 0.057 B + 0.012 C − 0.042 AC 0.8664 0.000 6.50 14.088

+ 0.046 A2 + 0.051 B2
Rs23 Rs23 = +4.65 − 1.00 A − 0.16 C + 0.28 A2 0.9406 0.000 4.61 33.672
tF2 tF2 = + 2.00 + 0.16 A + 0.061 B − 0.20 C 0.8186 0.001 5.12 18.270
tF3 tF3 = +1.75 + 0.16 A − 0.11 C 0.8476 0.000 4.00 20.209
tR3 tR3 = +6.81 − 1.12 C 0.8144 0.000 6.60 26.440
COF COF = +7.04 + 2.18 C 0.8249 0.000 12.05 27.381
a
  Only significant coefficients with P < 0.05 are included. Factors are in coded levels

were eliminated from the model through a backward
elimination process to obtain a simple and realistic model.
The adjusted R2 were well within the acceptable lim-
its of R2  ≥ 0.80 [23], which revealed that the experimen-
tal data show a good fit with the second-order polynomial
equations. For all the reduced models, P value of >0.05 is
obtained, implying that these models are significant. The
adequate precision values were found to be in the range of
14.08–33.67, which indicates an adequate signal, and there-
fore, the model is significant for the separation process. The
%CV for all the models was found to be <10 %, except for
COF (12.05 %). Therefore, the diagnostic plots (a) normal
probability plot of the residuals and (b) plot of residual ver-
sus predicted values [24] were analyzed for response COF.
Since the assumptions of normality and constant variance
of the residuals were found to be satisfied, the fitted model
for the response COF was accepted.
Response models (Table 2) suggest that among the fitted
models, k1 has the interaction term (AC) with largest abso-
lute coefficient (+0.042). The positive interaction between
A and C is statistically significant (P < 0.0001) for the k1
model. Perturbation plot was examined for response k1 to
understand the effect of an independent factor on a specific
response, with all other factors held constant at a reference
point [25]. It is evident from Fig. 2 that the response k1 was
highly influenced by the level of factor B followed by fac-
tor A. As expected, capacity factor k1 is least sensitive to
factor C.

13
Multiple Response Optimization of a HPLC Method
Fig. 2  Perturbation plot showing the effect of each of the independ-
ent factors on response k1, where A is %MeOH, B is %DEA and C is
mobile phase flow rate
Global Optimization of Multiple Responses
The targeted criteria for the optimization was to minimize
the resolution between the Ofloxacin enantiomeric pair,
reduce the tailing factors of enantiomers, minimize the
analysis time, increase the COF value as well as the reten-
tion factor k1. Global optimization of multiple responses
was achieved by employing desirability function computed
using JMP prediction profiler (Fig. 3). Desirability function
transfers the response variable to a 0–1 scale. A response
of 0 represents a completely undesirable response and 1
represents the most desirable response. Figure 3 depicts
that a smaller % of MeOH has positive impact on all the
factors except for the resolution Rs23 in which it leads to
excess resolution between the enantiomer pair, small %
of DEA showed a good impact on k1 value. However, its
effect on other factors was not significant as it showed
less curvature and the higher flow rate values showed sig-
nificant effect on k1, COF and tR3. The operating condi-
tions were chosen to achieve the maximum overall desir-
ability; all the responses were optimized simultaneously.
The optimum conditions were chosen by total desirability
as near as 1. The highest desirability value of 0.823 was
achieved at MeOH/hexane/MeCN-43.33/10/46.62 (v/v)
%, %AcOH/DEA-0.4/0.2 and flow rate as 1.4 mL min−1.
The predicted response values corresponding to the high-
est desirability value (0.823) were k1 = 0.61, Rs23 = 5.193,
tF2 = 1.68, tF3 = 1.57, tR3 = 6.14 min and COF = 8.25.
The prediction efficiency of the model was confirmed by
performing the experiment under the optimal condition,
and the corresponding chromatogram is shown in Fig. 4.
The average errors for k1, Rs23, tF2, tF3, tR3 and COF were
1.63, 0.05, −1.60, −3.95, −2.61 and 0.73 %, respectively,
within a difference of <4 %, indicating a good correlation
between the experimental and the predicted responses.
Validation of the Test Procedure
Linearity
The linearity of the proposed method was estimated by car-
rying out regression analysis at five concentration levels in
the range of 2–10 µg mL−1 for Ofloxacin (approximately
20–200 % of the nominal range of the analyte). The cali-
bration curve was obtained using the linear least squares
regression procedure. The representative linear equation
was y = 0.2375x − 0.0413 and y = 0.2356x − 0.0462 for
S-OFL and R-OFL, respectively. Correlation coefficients
were found to be more than 0.998 for both enantiomers.
The homoscedasticity for the calibration curves was veri-
fied by using Bartlett’s test and in that no statistical differ-
ence (P > 0.05) was found between the variances [26].
Limits of Detection and Quantitation
Calibration curves were plotted at five levels ranging from
0.05 to 1.0 % of the nominal analyte concentration [27].
The residual standard deviation of the response (σ) and
slope (s) of the calibration curve was used to calculate
the LOD as 3.3 σ/s and LOQ as 10 σ/s. Using the above
equations, the LOD and LOQ were estimated as 4.95 and
14.99 ng mL−1 for S-OFL, and 4.26 and 12.92 ng mL−1 for
R-enantiomer, respectively.
Specificity
Specificity of the method was evaluated by comparing the
chromatograms of placebo sample containing a mixture of
the commonly used excipients (starch, lactose monohy-
drate, methyl cellulose, titanium dioxide and magnesium
stearate) with that of the standard drugs. There were no
excipient peaks co-eluted with the analyte as well as with
IS, which shows that the developed method is selective and
specific to the excipients used in this study (Fig. 4).
Accuracy
Accuracy of the method was demonstrated by analyzing
quality control (QC) standards prepared at three levels of
80, 100 and 120 % of the expected assay value in the mar-
keted formulation. QC samples were prepared as three rep-
licates at each concentration level by spiking the standard
13

Fig. 3  Prediction profiler
obtained for the responses Rs23,
tF2, tF3, tR3, COF and k1
drugs with the placebo excipients, which were left over-
night to allow matrix–analyte interactions to occur, and then
analyzed as described in “Preparation of Sample Solution”
section. The % recovery of the enantiomers at each level
(n  = 3) and mean % recovery (n  = 9) were determined,
and data are presented in Table 3 where accuracy (%) was
expressed as [(calculated amount/predicted amount) × 100].
The recoveries of both enantiomers at each level were found
to lie within the acceptable criteria of the bias ±2 % [28].
The mean % recovery (n = 9) for each enantiomer was also
tested for significance by using Student’s t test, the null
13
V. Kannappan, S. S. Mannemala
hypothesis being that the recovery is unity or 100 %. Since
the calculated t-value (tCalc) for S-OFL (2.73) and R-OFL
(1.64) is less than the theoretical t-value (tCrit  = 4.30), at
5 % significance level, the null hypothesis was accepted.
These results indicate that the method is accurate, and there
was no interference from placebo in this study.
Precision
The precision was established by injecting three concen-
tration levels (2, 6 and 10 µg mL−1) for Ofloxacin with
Multiple Response Optimization of a HPLC Method

Fig. 4  Chromatograms cor-
responding to (a) placebo solu-
tion; b Levofloxacin (enantio-
pure); c a real sample of Glevo®
tablets containing Levofloxacin;
d a real sample of Zenoflox®
tablets containing Ofloxacin
enantiomers; e a synthetic
mixture of probenecid (IS) and
Ofloxacin enantiomers, under
optimum conditions

Table 3  Validation of method Validation parameters S-OFL R-OFL

for the determination of S-OFL
and R-OFL Concentration Results Concentration Results

Linearity (n = 6) 2–10 µg mL−1 y = 0.2375x − 0.0413 2–10 µg mL−1 y = 0.2356x − 0.0462

2
R  = 0.9988 R2 = 0.9981
LOD 4.95 ng mL−1 4.26 ng mL−1
−1
LOQ 14.99 ng mL 12.92 ng mL−1
Specificity The method is specific with respect to tablets ingredients (starch, lactose
monohydrate, methyl cellulose, titanium dioxide and magnesium stearate)
Accuracy (mean %recovery) (n = 3)
80 % W/W 98.78 80 % W/W 99.28
100 % W/W 99.63 100 % W/W 99.21
120 % W/W 99.47 120 % W/W 99.50
(mean %recovery, %CV) (n = 9) 99.29, 0.45 99.53, 0.49
Precision (%CV) (n = 6)
(a) Intraday precision 2.0 2.73 2.0 2.93
6.0 1.24 6.0 0.89
10.0 0.88 10.0 1.04
(b) Interday precision 2.0 1.84 2.0 2.73
6.0 1.39 6.0 0.87
10.0 0.70 10.0 0.91
Robustness (%assay, %CV)
%MeOH conc. (43.33 ± 0.5 %) 100.22, 0.53 99.84, 0.81
%DEA conc. (0.2 ± 2 %) 100.38, 0.25 100.05, 0.73

10 µg mL−1 of IS (probenecid) each in six replicates, for Robustness

intraday precision (repeatability) and on three consecutive
days for the intermediate precision (reproducibility). Pre-
cision was expressed by the RSD (%) of the analyte peak
area. Results for all studied compounds (Table 3) met the
proposed requirement %RSD ≤ 3 % [28].
To demonstrate the reliability of the developed method
for routine use, robustness of the proposed method was
evaluated with respect to small, but deliberate variations
in experimental parameters such as variations in %MeOH

13

concentration (43.38 % ± 0.5) and % DEA (0.2 ± 0.02)
did not alter the retention times, tailing factor (tF2) and res-
olution values more than 2 % in the proposed method. So,
it could be concluded that the developed method is robust.
Application of the Method
The proposed RP-HPLC method was applied to the quan-
titative analysis of the Ofloxacin and Levofloxacin tab-
lets. Ofloxacin enantiomers in real samples (Zenoflox
tablets) containing racemic Ofloxacin were analyzed by
the proposed method. Representative chromatograms are
presented in Fig. 4. The results achieved when analyzing
Zenoflox tablets were 100.07 (0.11) mg of Levofloxacin
and 99.95 (0.06) mg of R-OFL, respectively, with the val-
ues within parentheses being the %CV of the six replicates.
Assay of Levofloxacin was performed on Glevo-500 tab-
lets in which the presence of R-enantiomer is considered to
be an impurity. The result obtained for the assay of Levo-
floxacin was 499.6 (0.1) mg with the values within paren-
theses being the %CV of the six replicates. The content of
the R-OFL was not more than 0.5 %. Good agreement was
found between the assay results and the label claim of the
marketed formulations. The quantities found for Ofloxacin
as well as for Levofloxacin in tablets were in conformity
with the values claimed by the manufacturer.
Conclusions
In this paper, an efficient chiral HPLC method was devel-
oped, optimized and validated for the simultaneous esti-
mation of the Ofloxacin enantiomers in pharmaceutical
formulations and in bulk drugs using response surface
methodology. This method reduces overall analysis time
and provides essential information regarding the sensitiv-
ity of various chromatographic factors and their interaction
effects on separation attributes. Higher sensitivity, shorter
analysis time, use of polar non-aqueous organic solvents
and adequate resolution of the developed method dem-
onstrates that it can be extrapolated for semi-preparative
purpose and for LC–MS analysis of Ofloxacin enantiom-
ers. The proposed method was found to be linear, sensitive,
selective, precise and accurate. Therefore, it could be suc-
cessfully adopted for routine analysis of Ofloxacin enanti-
omers and in chiral impurity profiling of Levofloxacin in
bulk drugs and pharmaceutical formulations.
Acknowledgments  Sai Sandeep M is grateful to University Grants
Commission (UGC), New Delhi, India, for providing UGC BSR fel-
lowship and to UGC SAP- DRS Phase-I sponsored Department of
Pharmacy, Annamalai University, Tamil Nadu, India, for providing
the facilities to carry this research work.
13
V. Kannappan, S. S. Mannemala
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