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Chromatography Research International

Volume 2013, Article ID 432753, 5 pages
http://dx.doi.org/10.1155/2013/432753

Research Article
A Stability Indicating UPLC Method for the Determination
of Levofloxacin Hemihydrate in Pharmaceutical Dosage Form:
Application to Pharmaceutical Analysis

Batuk Dabhi,1 Bhavesh Parmar,1 Nitish Patel,1 Yashwantsinh Jadeja,1

Madhavi Patel,1 Hetal Jebaliya,1 Denish Karia,2 and A. K. Shah1
1
Department of Chemistry, Saurashtra University, Rajkot-360 005, Gujarat, India
2
Arts Commerce and Science College, Borsad 388540, Gujarat, India

Correspondence should be addressed to Batuk Dabhi; batukdabhi@gmail.com

Received 28 February 2013; Accepted 3 June 2013

Academic Editor: Antonio Martı́n-Esteban

Copyright © 2013 Batuk Dabhi et al. This is an open access article distributed under the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

A reliable and sensitive isocratic stability indicating RP-UPLC method has been developed and validated for quantitative analysis
and content uniformity study of levofloxacin hemihydrate in tablets. An isocratic method for analysis of levofloxacin hemihydrate
was archived on ACQUITY UPLC BEH C18 (100∗2.1) mm particle size 1.7 𝜇 columns within shorter runtime of 4 min with a
flow rate of 0.400 mL/min and using a photodiode array detector to monitor the eluate at 294 nm. The mobile phase consisted of
acetonitrile-buffer (23 : 77 v/v), (buffer: 20 mM K2 HPO4 + 1 mL triethylamine in 1 L water, pH = 2.50 by orthophosphoric acid).
Response was a liner function of drug concentration in the range of 0.5–80 𝜇g/mL (𝑟2 = 0.999) with a limit of detection and
quantification of 0.1 and 0.5 𝜇g/mL, respectively. Accuracy (recovery) was between 99.77% and 101.55%. The drug was subjected to
oxidation, hydrolysis, photolysis, and thermal degradation. Degradation products resulting from the stress studies did not interfere
with the detection of levofloxacin hemihydrate, and the assay is stability indicating.

1. Introduction available in the literature. There are no methods available for

quantitative analysis by UPLC. The method was developed
Levofloxacin hemihydrate (Figure 1) is a synthetic chemo- and validated as per ICH [11–13] and USP [14] guideline.
therapeutic antibiotic of the fluoroquinolone drug class and
is used to treat severe life-threatening bacterial infection
or bacterial infection that have failed to respond to other 2. Experimental
antibiotic classes.
IUPAC name is (S)-9-fluoro-2,3-dihydro-3-methyl-10- 2.1. Chemicals and Reagents. Levofloxacin hemihydrates ref-
(4-methylpiperazin-1-yl)-7-oxo-7H-pyrido[1,2,3-de]-1,4- erence standard (claim 99.48%) was provided by Cipla phar-
benzoxazine-6-carboxylic acid. maceuticals. Tablets (500 mg) of Levofloxacin were produced
Levofloxacin hemihydrate is highly water soluble in from a pharmacy. HPLC grade methanol and ortho phospho-
nature. It is also soluble in organic solvents, soluble in glacial ric acid were purchased from Merck India Limited, Mumbai,
acetic acid and chloroform, sparingly soluble in methanol, India. Analytical grade hydrochloric acid, sodium hydroxide
slightly soluble in ethanol, and practically insoluble in ether. pellets, and hydrogen peroxide solution 30% (v/v) were
Levofloxacin hemihydrate is odourless drug. Methods for obtained from Ranbaxy Fine Chemicals, New Delhi, India.
quantitative analysis of Levofloxacin by HPLC [1–7], by UV High purity water was obtained using Milli-Q (Millipore,
[8–10] spectroscopy in single as well as in combination, are Milford, MA, USA) water purification system.
2 Chromatography Research International

H was washed with water, and the combined filtrate was made
CH3 CH3 up to the mark with water (stock solution-B 500 𝜇g/mL),
O N transferred quantitatively 5 mL of above stock solution-B in
H3 C N N
50 mL volumetric flask and diluted up to the mark with
mobile phase (50 𝜇g/mL).

HO 2.5. Specificity/Force Degradation Study. Force degradation

F
studies were performed to evaluate the stability indicating
O O
properties and specificity of the method. All solutions used
in stress studies were prepared at an initial concentration of
Figure 1: Levofloxacin hemihydrate. 500 𝜇g/mL of levofloxacin hemihydrate and heat for 1 hr at
80∘ C. All samples were then diluted in mobile phase to give a
final concentration of 50 𝜇g/mL and filtered before injection.
0.16
0.12
(a.u.)

2.5.1. Acid Degradation Study. Acid decomposition was car-

0.08
0.04
ried out in 0.1 M HCl at a concentration of 500 𝜇g/mL of
0.00 levofloxacin hemihydrate and after heat for 1 hr at 80∘ C. The
0.20 0.60 1.00 1.40 1.80 2.20 2.60 3.00 3.40 3.80 stressed sample was cooled, neutralized, and diluted with
Minutes mobile phase.
Figure 2: Chromatogram of levofloxacin hemihydrates reference
standard. 2.5.2. Base Degradation Study. Base decomposition was car-
ried out using 0.1 M NaOH at a concentration of 500 𝜇g/mL
and heat for 1 hr at 80∘ C. After cooling, the solution was
neutralized and diluted with mobile phase.
2.2. Instruments and Condition. The fast liquid chromatog-
raphy was performed using waters UPLC system with photo-
diode array detector. Chromatogram and data were recorded 2.5.3. Oxidative Degradation. Oxidation solutions for oxida-
by means of Empower software. The chromatographic system tive stress studies were prepared using 3% H2 O2 at a concen-
was performed using an Acquity BEH C18 (100 × 2.1 mm) tration of 500 𝜇g/mL of levofloxacin and after heat for 1 hr at
id., 1.7 𝜇m column. Separation was achieved using a mobile 80∘ C. The sample solution was cooled and diluted accordingly
phase consisted of buffer : acetonitrile (77 : 23 v/v) (buffer: with the mobile phase.
20 mM KH2 PO4 + 1 mL triethylamine in 1 litre water, pH
= 2.5 adjust with orthophosphoric acid) at a flow rate of 2.5.4. Thermal Degradation. For thermal stress testing, the
0.4 mL/min in only 4 minute runtime. The column tempera- drug solution 500 𝜇g/mL was heated for 1 hr at 80∘ C, cooled,
ture was maintained at 30∘ C, injection volume was 2 𝜇L, and and used for the study.
detection wavelength was set at 294 nm for determination of
levofloxacin hemihydrate (Figure 2). 2.5.5. Photo Degradation. For Photolytic degradation, the
powder drug has been exposed to sunlight for 48 hrs and used
2.3. Preparation of Standard Solution. The stock solution for the study.
of levofloxacin hemihydrate (500 𝜇g/mL) was prepared by
accurately weighing 25 mg of levofloxacin hemihydrate refer- 3. Method Validation
ence standard and transferring to 50 mL standard volumetric
flask containing approximately 25 mL of water. The flask was The method was validated as per ICH guideline. The method
sonicated for 10 minutes to dissolve the solids. Volumes were was validated by performing system suitability, linearity, limit
made up to the mark with water (500 𝜇g/mL stock solution- of Detection (LOD), limit of quantification (LOQ), precision,
A). Transferred 5 mL of above stock solution-A to a 50 mL accuracy, selectivity, and robustness.
volumetric flask and diluted up to the mark with mobile phase
to obtained working standard solution (50 𝜇g/mL) of drug. 3.1. Precision. Method precision of the analytical method
was determined by analyzing six sets of sample solution
2.4. Preparation of Sample Solution. For analysis of the tablet preparation. Assay of all six replicate sample preparations was
dosage form, ten tablets were weighed individually, and their determined, and mean percentage of assay value, standard
average weight was determined. The tablets were crushed deviation and percentage of relative standard deviation for
to fine homogenous powder, and a quantity equivalent to the same were calculated. Intermediate precision of the
25 mg levofloxacin was transferred in a 50 mL volumetric analytical method was determined by performing method
flask. Added about 25 mL of water to the flask, shaken precision on another day by another analyst using different
for 10 minutes, and then sonicated for 15 minutes. The make of raw materials under same experimental condition.
solution was allowed to stand at room temperature and Overall assay value of method precision and intermediate
filtered through Whatman no. 41 filter paper. The residue precision was compared and percentage of difference and
Chromatography Research International 3

overall percentage of relative standard deviation were calcu- Table 1: Result of precision, linearity, LOD, and LOQ study.
lated.
Parameter UPLC
Linearity (𝜇g/mL) 0.5–80
3.2. LOD and LOQ. Sensitivity of the method was deter-
mined by establishing the LOD and LOQ. The LOD and Correlation coefficient (𝑟2 ) 0.9994
LOQ were established at signal-to-noise ratio of 3 : 1 and 10 : 1, Slope (m) 46965
respectively. Intercept (c) 85486
LOD (𝜇g/mL) 0.1
3.3. Linearity. Linearity test solutions for the assay method LOQ (𝜇g/mL) 0.5
were prepared from a stock solution at 7 concentration levels Accuracy (% RSD) 0.21–0.76
of the assay analyte concentration (20, 30, 40, 50, 60, 70, Interday precision (RSD, 𝑛 = 6) 0.57
and 80 𝜇g/mL). The peak area versus concentration data was
Intraday precision (RSD, 𝑛 = 6) 0.26
analyzed with least squares linear regression. The slope and
𝑌-intercept of the calibration curve were reported.
established by determining the overall (inter-day and intra-
3.4. Accuracy. Accuracy of the developed method was con- day) method precision for assay. For intermediate precision,
firmed by doing recovery study as per ICH guidelines at overall assay value (𝑛 = 12) was 101.24% and RSD = 0.36%.
three different concentration levels 50%, 100%, and 150% Percentage of RSD value less than 2% indicates that this
by replicate analysis (𝑛 = 3). The results indicated that method is highly precise.
the method is highly accurate for the determination of
levofloxacin hemihydrate. 4.1.2. LOD and LOQ. All the results of LOD and LOQ
data were within the acceptance criteria; hence, it can be
3.5. Robustness. The robustness of the assay method was concluded that the LOD and LOQ of the method were
established by introducing small changes in the chromato- 0.1 𝜇g/mL and 0.5 𝜇g/mL, respectively. The signal-to-noise
graphic condition which included percentage of methanol ratio for the LOD was well within the acceptance criteria
in mobile phase (58% and 62%), flow rate (0.38 and which means more than 3.3, and for the LOQ it was more than
0.42 mL/min), and column oven temperature (25∘ C and 10.0. Furthermore, the data of linearity extension chart up to
35∘ C). Robustness of the method was studied using six LOQ level also suggest that the levofloxacin can be quantified
replicates at a concentration level of 50 𝜇g/mL of levofloxacin up to 0.5 𝜇g/mL with well precisely and accurately.
hemihydrate.
4.1.3. Linearity. The calibration curve for the levofloxacin
3.6. Solution Stability. The solution stability of levofloxacin hemihydrate was linear over the concentration range of 0.5–
hemihydrate in the assay method was carried out by leaving 80 𝜇g/mL. The data for the peak area versus concentration
both the sample and reference standard solutions in tightly were treated by linear regression analysis, and the correlation
capped volumetric flasks at room temperature for 48 hr. The coefficient (𝑟2 ) was obtained (0.9994). The regression equa-
same sample solution was assayed at 6 hr intervals over the tion for the calibration curve was found to be 𝑦 = 46, 965𝑥 +
study period. The percentage of RSD of the levofloxacin 85, 486 (Table 1).
hemihydrate assay was calculated for solution stability exper-
iments. An additional study was carried out using the stock 4.1.4. Accuracy. The percentage recovery of levofloxacin
solution by storing it in a tightly capped volumetric flask at hemihydrate in the drug tablets samples was obtained in a
4∘ C. range from 99.97% to 101.55%, respectively (Table 2). Percent-
age of RSD value of replicated sets was less than 2% which
indicates that this method is highly accurate.
4. Result and Discussion
4.1. Method Validation 4.1.5. Robustness. The robustness of an analytical procedure
refers to its ability to remain unaffected by small and delib-
4.1.1. Precision. The precision of the method was determined erate variations in method parameters and provides an indi-
by repeatability (intraday precision) and intermediate pre- cation of its reliability for routine analysis. The robustness of
cision (interday precision) of the levofloxacin hemihydrate the method was evaluated by assaying the same sample under
standard solution. The precision of the assay method was different analytical conditions deliberately changing from the
evaluated by carrying out six independent assays. For assay original condition. The results obtained from assay of the test
method (𝑛 = 6), percentage of RSD for system precision was solutions were not affected by varying the conditions and
0.45% on the same day (intra-day) and 0.41% on different were in accordance with the results for original conditions
days (inter-day). The mean values of method precision (Table 3). The percentage of RSD value of assay determined
(repeatability) were 101.2% (RSD = 0.26%) for assay on the for the same sample under original conditions and robustness
same day (intra-day) and 101.58% (RSD 0.57%) for assay conditions was less than 2.0%, indicating that the developed
on different days (inter day). Intermediate precision was method was robust.
4 Chromatography Research International

Table 2: Accuracy study.

Level % No. Amount of drug added (𝜇g/mL) Amount of drug found (𝜇g/mL) Recovery (%) Mean recovery (%) RSD (%)
1 25.23 25.15 99.87
50 2 25.29 25.64 101.40 100.63 0.76
3 25.34 25.63 100.62
1 50.82 50.98 100.32
100 2 50.60 50.40 99.62 99.97 0.35
3 50.27 50.52 99.97
1 75.21 76.21 101.34
150 2 75.39 76.72 101.77 101.55 0.21
3 75.06 76.61 101.54

Table 3: Robustness study.

System suitability data

Conditions Assay (%) RT (min)
Theoretical plates Asymmetry
Flow mL/min
0.38 98.89 1.27 5244 1.14
0.40 99.08 1.24 5253 1.11
0.42 99.21 1.25 5276 1.10
Column temperature
25∘ C 98.93 1.46 5378 1.12
30∘ C 99.08 1.24 5253 1.11
35∘ C 99.19 1.14 5182 1.08
ACN: buffer ratio
21 : 79 V/V 99.87 1.40 5402 1.10
23 : 77 V/V 99.08 1.24 5253 1.11
25 : 75 V/V 100.27 1.16 5185 1.13

Table 4: Specificity Study. 0.10

Stress condition Time Purity Purity % Degradation 0.08

angle threshold
0.06
(a.u.)

Acid Hydrolysis 1 hr 0.409 0.874 8.12

Base Hydrolysis 1 hr 12.38 0.04
1.107 13.28
Oxidation 1 hr 4.578 12.82 66.44 0.02
Thermal 1 hr 0.854 1.057 24.37 0.00
Photo 5 days 0.748 1.682 27.82 0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00
Minutes

Figure 3: H2 O2 degradation study of levofloxacin.

4.1.6. Solution Stability. The percentage of RSD of the assay
of levofloxacin hemihydrate from the solution stability exper-
iments was within 2%. The results of solution stability
experiments confirm that the sample solutions used during separation of levofloxacin peak from the degraded samples
the assay were stable up to 48 hr at room temperature and up derived from the empower software. The degradation of lev-
to 8 days at 4∘ C. ofloxacin hemihydrate was found to be very similar for both
the tablets and standard. Typical chromatograms obtained
4.1.7. Specificity. The specificity of the developed method following the assay of stressed samples are shown in Figures
was determined by injecting sample solutions (50 𝜇g/mL) 3, 4, and 5.
which were prepared by forcibly degrading under such stress Levofloxacin hemihydrate standard and tablet powder
conditions as heat, light, oxidative agent, acid, and base were found to be quite stable under light and heat conditions.
under proposed chromatographic condition. The stability A slight decomposition was seen on exposure of levofloxacin
indicating capability of the method was established from the drug solution to acid and heat. On the other hand, the drug
Chromatography Research International 5

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