Supplementary Information
HPLC/UV method development
Mobile phase was prepared by using acetonitrile and distilled water in a ratio of 14:86. The
orthophosphoric acid was used to adjust the pH at 3.0. Mobile phase was filtered with 0.45 µ
nylon filter and degassed using an ultrasonic bath.
Pure ofloxacin (100 mg) was accurately weighed and dissolved in mobile phase (100 mL) to
prepare standard solution (1000 ppm) of pure ofloxacin. This standard solution was then diluted
to make different concentrations. Similarly, different concentrations of ofloxacin prodrugs (2 and
6) were prepared by dissolving accurately weighed amount in mobile phase and then by diluting
it. All these solutions were filtered through 0.45 µ nylon syringe filter before packing them into
HPLC vials.
A Shim-Pack ODS 5μm (4.6 × 250 mm) column was selected for optimum chromatographic
separation. Mobile phase comprising of acetonitrile and distilled water (14:86, pH 3.0) was used
in isocratic mode, the flow rate was kept 1.0 mL min-1 and injection volume was set at 20 μL.
The chromatographic separations were detected at 294.5 nm.
HPLC/UV method validation
The ICH guidelines were followed to determine the validation of the developed method under
the chromatographic conditions discussed above. A range of standard solutions (5-50 μg/mL) of
HPC-ofloxacin conjugate 2 and HEC-ofloxacin conjugate 6 were prepared to check the linearity
of the developed method for both prodrugs. The chromatograms were obtained after injecting 20
μL of each standard solution and separate calibration curves were constructed for both prodrugs
by plotting peak areas vs. concentrations. The r2 values were determined by linear regression
method and found to be 0.9996 and 0.9991 for HPC-ofloxacin conjugate 2 and HEC-ofloxacin
conjugate 6, respectively.
A standard solution containing 10 μg/mL of HPC-ofloxacin conjugate 2 was injected six
times in the HPLC system to check the precision of the developed method and relative standard
deviation (RSD) was determined. The repeatability RSD value was found to be 0.325%. The
solution was analyzed on the same day (intra-day precision) and on three different days (interday precision) to determine the variability of the method. The RSD values were found 0.651%
and 0.412% for intra-day precision and inter-day precision, respectively. Different samples were
spiked with known amount of the HPC-ofloxacin conjugate to determine the accuracy or %
recovery of the method. The %age recovery was calculated by the amounts of samples
recovered. The % recovery was found to be 99.27%. All these parameters were also determined
for HEC-ofloxacin conjugate 6.
For HEC-ofloxacin conjugate 6, the repeatability RSD value was found to be 0.446%. The
intra-day precision and inter-day precision RSD values were calculated as 0.783% and 0.367%,
respectively, whereas % recovery was found to be 98.19%. The HPLC/UV method validation
parameters are summarized in Table S1.
Specificity of the method was evaluated by analyzing a blank solution and a standard
solution (10 μg/mL) under the optimized chromatographic conditions.
The obtained
chromatograms showed no interference by any impurity and optimum retention time was
observed for the drug samples which confirmed the specificity of the method. Robustness of the
method was confirmed by slightly changing the composition of the mobile phase, pH of the
mobile phase and flow rate of the mobile phase and no significant change was observed.
The LOD was calcd. from the linearity curve by applying the formula (LOD = 3S/D). The LOQ
of the method was found to be about thrice of the LOD.
Table S1. Validation Parameters of HPC- and HEC-ofloxacin conjugates
No.
Parameter
HPC- ofloxacin
HEC- ofloxacin
conjugate 2
conjugate 6
(Mean)
(Mean)
1
Concentration range
5-50 μg mL-1
5-50 μg mL-1
2
LOD
25 ng mL-1
25 ng mL-1
3
LOQ
75 ng mL-1
75 ng mL-1
4
Linear regression co-efficient (r2)
0.9996
0.9991
5
Precision Intra-day (RSD)*
0.651%
0.783%
6
Precision Inter-day (RSD)*
0.412%
0.367%
7
Accuracy (% recovery)*
99.27%
98.19%
8
Repeatability (RSD)*
0.325%
0.446%
*Each value is mean of six observations
The blood plasma levels at different times after the administration of pure ofloxacin, physical
mixtures of ofloxacin with HPC and HEC, HPC-ofloxacin conjugate 2 and HEC-ofloxacin
conjugate 6 are summarized in Table S2.
Table S2. Comparison of (the mean ± SEM) plasma concentration vs. time profile after oral
administration of pure ofloxacin, physical mixture of ofloxacin with HPC and HEC, ofloxacin
prodrugs 2 and 6 (35 and 39 mg respectively) equivalent to pure ofloxacin (14 mg) to 30 rabbits
Sample Time
Control 1a
Control 2b
Control 3 c
HPC-ofloxacin
HEC-ofloxacin
No.
(h)
Conc. (μg/mL)
Conc. (μg/mL)
Conc. (μg/mL)
Conc. (μg/mL)
Conc. (μg/mL)
1
0.17
1.21 ± 0.25
1.03 ± 0.31
0.88 ± 0.20
0.79 ± 0.15
0.45 ± 0.21
2
0.5
2.57 ± 0.24
2.39 ± 0.29
2.11 ± 0.23
1.05 ± 0.14
0.71 ± 0.18
3
1.0
3.75 ± 0.25
3.07 ± 0.19
2.94 ± 0.28
1.61 ± 0.15
1.31 ± 0.11
4
1.5
2.89 ± 0.25
3.49 ± 0.27
3.43 ± 0.26
2.10 ± 0.23
1.93 ± 0.19
5
2
2.11 ± 0.26
2.19 ± 0.31
2.34 ± 0.22
2.61 ± 0.17
2.49 ± 0.12
6
4
1.40 ± 0.24
1.48 ± 0.18
1.53 ± 0.27
3.56 ± 0.14
3.67 ± 0.30
7
6
0.99 ± 0.33
1.09 ± 0.22
1.14 ± 0.29
2.98 ± 0.13
3.26 ± 0.22
8
8
0.65 ± 0.26
0.83 ± 0.28
0.91 ± 0.19
2.21 ± 0.11
2.78 ± 0.19
9
12
0.44 ± 0.20
0.54 ± 0.27
0.59 ± 0.32
1.43 ± 0.12
2.01 ± 0.16
10
24
0.30 ± 0.23
0.25 ± 0.20
0.31 ± 0.19
0.93 ± 0.19
1.28 ± 0.09
11
36
0.18 ± 0.19
0.16 ± 0.17
0.18 ± 0.19
0.51 ± 0.10
0.88 ± 0.09
a
Pure ofloxacin; b Physical mixture of ofloxacin with HPC; c Physical mixture of ofloxacin with
HEC