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Asian J. Pharm. Res. 2011; Vol. 1: Issue 4, Pg 119-125
[AJPRes.]
ISSN- 2231–5683 (Print)
www.asianpharmaonline.org
ISSN- 2231–5691 (Online) 0974-3618
RESEARCH ARTICLE
Rapid RP-HPLC Method for Simultaneous Estimation of Sparfloxacin,
Gatifloxacin, Metronidazole and Tinidazole
Mahmoud M. Sebaiy 1 , Abdullah A. El-Shanawany 1 , Sobhy M. El-Adl 1 , Lobna M. Abdel-Aziz 1
and Hisham A. Hashem 2 .
1 Medicinal Chemistry Department, Faculty of Pharmacy, Zagazig University, Egypt.
2 Analytical Chemistry Department, Faculty of Pharmacy, Zagazig University, Egypt.
*Corresponding Author E-mail: sebaiy_pharma@yahoo.com
ABSTRACT:
An isocratic RP-HPLC method had been developed for rapid simultaneous separation and determination of
sparfloxacin, gatifloxacin, metronidazole and tinidazole in pure form or in presence of some impurities within 5
minutes. Separation was carried out on a Chromolith ® Performance RP-18e (100 x 4.6 mm) using a mobile phase of
MeOH : 0.025M KH 2 PO 4 adjusted to pH 3 using ortho - phosphoric acid (20:80, v/v) at ambient temperature. The
flow rate was 4 ml/min and maximum absorption was measured at 290 nm. The standard curve was linear in the
concentration range of 1-80 µg/mL for all drugs. The retention time of sparfloxacin, gatifloxacin, metronidazole and
tinidazole was noted to be 4.3, 3, 1.8 and 1.2 minutes respectively, indicating shorter analysis time. The method was
validated according to ICH guidelines. The proposed method was found to be accurate, reproducible, and consistent. It
was successfully applied for the analysis of these drugs in marketed formulations and could be effectively used for the
routine analysis of formulations containing any one of the above drugs, or a combination, without any alteration in the
chromatographic conditions.
KEY WORDS: RP-HPLC; Sparfloxacin; Gatifloxacin; Metronidazole; Tinidazole.
1. INTRODUCTION:
Fluoroquinolones are a class of compounds that comprise
a large and expanding group of synthetic antimicrobial
agents. Structurally, all fluoroquinolones contain a fluorine
atom at the 6-position of the basic quinolone nucleus.
Despite the basic similarity in the core structure of these
molecules, their physicochemical properties,
pharmacokinetic characteristics and microbial activities
can vary markedly across compounds 1 .
Quinolones act by inhibiting the activities of DNA gyrase
(enzyme catalyzing changes in the degree of doublestranded
DNA supercoiling) in gram-negative bacteria,
which in turn inhibit replication and transcription of
bacterial DNA. Prevention of DNA synthesis ultimately
results in rapid cell death. This unique mechanism of action
may account for the low rate of cross-resistance with
other antimicrobial classes 2 .
Received on 10.07.2011 Accepted on 14.08.2011
© Asian Pharma Press All Right Reserved
Asian J. Pharm. Res. 1(4): Oct. - Dec. 2011; Page 119-125
119
Quinolones similarly inhibit the in vitro activities of DNA
topoisomerase IV (enzyme mediating relaxation of duplex
DNA and the unlinking of daughter chromosomes
following replication) which is believed to be the
primary target in gram-positive bacteria 3 .
Sparfloxacin (5-Amino-1-cyclopropyl-7-(cis-3,5-dimethyl-
1-piperazinyl) -6,8- difluoro-1,4 dihydro-4-oxo-3-
quinolinecarboxylic acid) and Gatifloxacin ((±)-1-
cyclopropyl-6-fluoro-1,4-dihydro-8-methoxy-7-(3-methyl-
1-piperazinyl)-4-oxo-3-quinolinecarboxylic acid) are
fluoroquinolones and antimicrobials with potent activity
against a broad spectrum of bacteria.
Metronidazole {2-methyl-5-nitroimidazole-1-ethanol} and
Tinidazole {1-(2-ethyl-sulphonyl ethyl) -2-methyl-5-
nitroimidazole} are used as antiamoebic, antiprotozoal and
antibacterial agents 4 .
Some HPLC methods had been developed for determination
of these drugs individually 5-9 or in combination with other
drugs 10-13 but No HPLC method for simultaneous estimation
of these four drugs using monolithic silica columns has
been reported till date.

Asian J. Pharm. Res. 2011; Vol. 1: Issue 4, Pg 119-125
In the present study, an attempt has been made to develop a
method for the simultaneous estimation of sparfloxacin,
gatifloxacin, metronidazole and tinidazole. It can also be
applied for routine analysis of either one or of any
combinations of these drugs in dosage forms.
2. EXPERIMENTAL:
2.1. Apparatus:
• Waters 2487 ® HPLC instrument (U.S.A) with Waters
automated gradient controller, Chromolith ® Performance
RP-18e column (100 x 4.6 mm), dual absorbance detector,
binary 515 HPLC pumps and connected to PC computer
loaded with Millenium 32 software.
• Consort P400 ® digital pH-meter for pH adjustment.
2.2. Materials and reagents:
• All solvents and reagents were of an HPLC analytical
grade (methanol, potassium dihydrogen phosphate and
ortho - phosphoric acid were supported from Romil,
England).
• Sparfloxacin (Global Napi), Gatifloxacin (EPCI),
Metronidazole (Sanovi Aventis) and Tinidazole (Medical
Union of Pharmaceuticals). Standard solutions 400 µg.ml -1
were prepared individually by dissolving 40 mg of each
pure drug in 100 ml of the mobile phase.
• Mobile phase was a freshly prepared binary mixture of
methanol: 0.025M potassium dihydrogen phosphate
adjusted to pH 3 using ortho - phosphoric acid (20:80, v/v),
filtered and degassed using 0.45µm membrane filter.
• Manufacturing impurities like Benzyl amine,
Ethylene diamine, 2,3,4-trifluoroaniline, 2,6-dimethyl
piperazine, Diethyl malonate and 2-methyl imidazole were
supported from Merck, Germany.
2.3. Pharmaceutical preparations:
The following available pharmaceutical preparations were
analyzed
• Spara ® tablets labeled to contain 200 mg sparfloxaacin
per tablet. Batch No. 911601 (Global Napi, Egypt).
• Gatiflox ® tablets labeled to contain 400 mg
gatifloxacin per tablet. Batch No. 171080310 (EPCI,
Egypt).
• Flagyl ® tablets labeled to contain 500 mg
metronidazole per tablet. Batch No. 10E70 (Sanovi
Aventis, Egypt).
• Protozole ® tablets labeled to contain 500 mg tinidazole
per tablet. Batch No. 90133 (MUP, Egypt).
[AJPRes.]
2.4.2. Sample preparation:
10 tablets of each formulation were weighed and powdered.
An accurately amounts of the powder equivalent to 40 mg
of each drug were dissolved in 25 ml of the mobile phase,
filtered into 100 - ml measuring flask and completed to
volume with the mobile phase. The procedure was then
completed as mentioned above under the general procedure.
3. RESULTS AND DISCUSSION:
Monolithic silica columns were first introduced in 1991 by
Minakuchi and Soga (14) . The preparation of these silica rod
materials involved a sol-gel process using highly pure
silica. The formed silica rod is then encased in poly ether
ethyl ketone shrink-warp tubing, which prevents void
formation. The obtained highly porous skeleton is
characterized by a bimodal pore structure consisting of
large macropores (diameter 2 µm) and mesopores (13 nm in
diameter). The large macropores are responsible for a low
flow resistance and therefore allow for the application of
high eluent flow rates, while the small pores ensure
sufficient surface area (300 m 2 /g approximately) for
separation efficiency. As aresult, High flow rates could be
used with monolithic columns due to the high porosity of
the column provided mainly with macropores. Besides, high
efficiency is ensured by the mesopores that provide very
large surface area for separation 15 (Fig. 1).
A
2.4. Procedures:
2.4.1. Preparation of calibration curves:
Appropriate mixed dilutions of the standard stock solutions
of sparfloxacin, gatifloxacin, metronidazole and tinidazole
were done in 10 - ml volumetric flasks to get a final
concentrations of 1, 10, 20, 40, 60 and 80 µg.ml -1 for all
drugs. A 10 l of each mixture was injected into the column
and the chromatogram was obtained at 290 nm. A graph
was plotted as concentration of drugs against response
(peak area) and it was found to be linear for all drugs.
120
B
Fig. (1) Monolithic Silica Skeleton A, Macropores and Mesopores
B.

Asian J. Pharm. Res. 2011; Vol. 1: Issue 4, Pg 119-125
The difference between monolithic and conventional
particle-packed columns is shown in Figure 2.
Conventional Silica "Particle-Based"
High flow resistance:
Limits ability to shorten run times.
High backpressure:
Reduces life of system.
[AJPRes.]
to blockage and long column life time are also advantages
of high porosity 16 .
3.1. Optimization of Chromatographic Conditions:
All chromatographic conditions are illustrated in table 1.
Spectroscopic analysis of the drugs showed that
sparfloxacin, gatifloxacin, metronidazole and tinidazole
have maximum UV absorbance ( max ) at 291 nm, 290 nm,
300 nm and 298 nm respectively. Therefore, the
chromatographic detection was performed at 290 nm using
a UV – Visible detector. The method was performed on a
Chromolith ® Performance RP-18e (100 x 4.6 mm)
supported from Germany. Furthermore, It was observed that
the optimized mobile phase was determined as a mixture of
methanol: 0.025M potassium dihydrogen phosphate
adjusted to pH 3 using ortho - phosphoric acid (20:80, v/v)
at a flow rate of 4 ml/min. Under these conditions,
sparfloxacin, gatifloxacin, metronidazole and tinidazole
were eluted at 4.3, 3, 1.8, and 1.2 minutes respectively with
a run time of 10 minutes. A typical chromatogram for
simultaneous estimation of these drugs obtained by using
the aforementioned mobile phase is illustrated in figures 3
(authentic mixture) and 4 (tablet formulations).
Monolithic porous silica rod
High flow rates:
Significantly shorter run times.
Low backpressures:
Less stress on system.
Fig.(2) Representative conventional particle-packed vs. monolithic
silica HPLC columns.
Furthermore, the separation efficiency of monolithic
columns does not decrease significantly when the flow rate
is increased as in case of particulate columns. Accordingly,
it is possible to operate monolithic columns at high flow
rates with minimal loss of peak resolution. High resistance
Fig.(3) HPLC Chromatogram of authentic mixture of sparfloxacin
(s), gatifloxacin (g) , metronidazole (r) and tinidazole (t).
Column : Chromolith ® Performance RP-18e (100 x 4.6 mm).
Mobile phase : MeOH : 0.025M KH 2PO 4 adjusted to pH 3 using
ortho phosphoric acid (20:80, v/v).
Flow rate : 4 ml/min.
pH : 3.
121

Asian J. Pharm. Res. 2011; Vol. 1: Issue 4, Pg 119-125
[AJPRes.]
Table(1). Chromatographic Conditions for the proposed methods.
Parameters
Conditions
Column
Chromolith ® Performance RP-18e (100 x 4.6 mm)
Mobile phase
UV detection, nm 290
Flow rate, ml/min 4
Injected volume, µl 10
Pressure, psig 2980
Temperature
Ambient
Isocratic binary mobile phase of MeOH : 0.025M KH 2PO 4 adjusted to pH 3 using ortho - phosphoric acid
(20:80, v/v), filtered and degassed using 0.45µm membrane filter
Table(2). Results of the analysis for the proposed methods.
parameters Sparfloxacin* Gatifloxacin* Metronidazole * Tinidazole*
Taken
µg/ml
Found
µg/ml
Recovery
%
Taken
µg/ml
Found
µg/ml
Recovery
%
Taken
µg/ml
Found
µg/ml
Recovery
%
Taken
µg/ml
Found
µg/ml
Recovery
%
1 1.006 100.63 1 1.002 100.25 1 1.018 101.87 1 0.996 99.61
10 9.94 99.41 10 9.99 99.96 10 10.04 100.42 10 10.13 101.34
20 19.90 99.52 20 20.30 101.52 20 20.07 100.35 20 20.03 100.15
40 39.83 99.56 40 39.92 99.81 40 39.59 98.97 40 39.64 99.09
60 60.45 100.75 60 60.22 100.36 60 59.78 99.63 60 60.33 100.55
80 81.10 101.37 80 80.14 100.17 80 80.34 100.43 80 79.91 99.88
Mean 100.21 100.34 100.28 100.10
±SD 0.821 0.610 0.970 0.781
±RSD 0.820 0.607 0.967 0.780
±SE 0.335 0.250 0.396 0.319
Variance 0.675 0.456 0.940 0.610
Slope 12976 15527 8529.4 6282.1
L.D. 0.250 0.250 0.300 0.300
L.Q. 0.750 0.750 0.900 0.900
S.S. 7 x 10 -8 5 x 10 -8 1 x 10 -7 2 x 10 -7
* Average of three independent procedures.
Fig.(5) HPLC Chromatogram of authentic sparfloxacin (s) in presence
Fig.(4) HPLC Chromatogram of drugs in mixture of Spara ® , Gatiflox ® ,
of benzyl amine (ba), 2,6-dimethyl piperazine (dmp) and diethyl
Flagyl ® and Protozole ® tablet formulations.
malonate (dem).
Column : Chromolith ® Performance RP-18e (100 x 4.6 mm).
Column : Chromolith ® Performance RP-18e (100 x 4.6 mm).
Mobile phase : MeOH : 0.025M KH 2PO 4 adjusted to pH 3 using
Mobile phase : MeOH : 0.025M KH 2PO 4 adjusted to pH 3 using
ortho phosphoric acid (20:80, v/v).
ortho phosphoric acid (20:80, v/v).
Flow rate : 4 ml/min.
Flow rate : 4 ml/min.
pH : 3.
pH : 3.
122

Asian J. Pharm. Res. 2011; Vol. 1: Issue 4, Pg 119-125
[AJPRes.]
Also, chromatographic conditions were appropriate for
separation of each drug from its manufacturing impurities.
Benzyl amine, 2,6-dimethyl piperazine and diethyl
malonate 17 can be separated from sparfloxacin and eluted at
0.47, 1,38 and 6.25 minutes, respectively (fig. 5). Ethylene
diamine and 2,3,4-trifluoroaniline 18 can be separated from
gatifloxacin and eluted at 0.53 and 1.19 minutes,
respectively (fig. 6). 2-methyl imidazole (19) can be separated
from tinidazole and metronidazole and eluted at 0.86
minute (fig. 7).
Fig.(6) HPLC Chromatogram of authentic gatifloxacin (g) in presence
of ethylene diamine (e) and 2,3,4-trifluoroaniline (f).
Column : Chromolith ® Performance RP-18e (100 x 4.6 mm).
Mobile phase : MeOH : 0.025M KH 2PO 4 adjusted to pH 3 using
ortho phosphoric acid (20:80, v/v).
Flow rate : 4 ml/min.
pH : 3.
3.2. Method Validation:
The developed methods were validated according to
international conference of harmonization guidelines 20 .
123
Fig.(7) HPLC Chromatogram of authentic tinidazole (t) and
metronidazole (r) in presence of 2-methyl imidazole (mi).
Column : Chromolith ® Performance RP-18e (100 x 4.6 mm).
Mobile phase : MeOH : 0.025M KH 2PO 4 adjusted to pH 3 using
ortho phosphoric acid (20:80, v/v).
Flow rate : 4 ml/min.
pH : 3.
3.2.1. Linearity:
Six different concentrations of a mixture of all drugs in both
methods were prepared for linearity studies. The response
was measured as peak area. The calibration curves obtained
by plotting peak area against concentration showed linearity
in the concentration range of 1 - 80 µg.ml -1 for all drugs.
Linear regression equation of sparfloxacin, gatifloxacin,
metronidazole and tinidazole was found to be y = 12976x +
13078, y = 15527x + 22871, y = 8529.4x – 567.18 and y =
6282.1x + 273.23 respectively and the regression
coefficient values (r) were found to be 0.9991, 0.9992,
0.9999 and 0.9995 respectively indicating a high degree of
linearity for all drugs.

Asian J. Pharm. Res. 2011; Vol. 1: Issue 4, Pg 119-125
[AJPRes.]
3.2.2. Accuracy:
The accuracy of the methods was determined by
investigating the recovery of drugs at concentration levels
covering the specified range (three replicates of each
concentration). The results showed excellent recoveries
(table 2).
3.2.3. precision:
Intraday precision was evaluated by calculating standard
deviation (SD) of five replicate determinations using the
same solution containing pure drug. The SD values revealed
the high precision of the methods (values vary from 0.78 to
0.96). For inter - day reproducibility on a day - to - day
basis, a series was run, in which the standard drug solutions
were analyzed each for five days. The day - to - day SD
values were in the range of 0.98 - 1.9.
3.2.4. Specificity:
The specificity studies revealed the absence of any excipent
or impurity interference, since none of the peaks appeared
at the same retention time of sparfloxacin, gatifloxacin,
metronidazole and tinidazole as shown in figures 5, 6 and
7.
3.2.5. L.D. and L.Q.:
For determining the limit of detection (L.D.) and limit of
quantitation (L.Q.), the method based on signal – to - noise
ratio (3:1 for L.D. & 10:1 for L.Q.) was adopted. The limit
of detection for both sparfloxacin and gatifloxacin was
0.250 µg.ml -1 and for both metronidazole and tinidazole was
0.300 µg.ml -1 while the limit of quantitation for both
sparfloxacin and gatifloxacin was 0.750 µg.ml -1 and for
both metronidazole and tinidazole was 0.900 µg.ml -1
(table 2).
3.2.6. Robustness:
The robustness of the methods was evaluated by making
small changes in the flow rate (3.9, 4, 4.1), pH of mobile
phase within a range of ± 0.2 unit of the optimized pH and
mobile phase ratio keeping the other chromatographic
conditions constant where the effect of the changes was
studied on the percent recovery of drugs. The changes had
negligible influence on the results as revealed by small SD
values ( 1.93).
3.2.7. Applications:
Some Pharmaceutical formulations containing stated drugs
in combination and in pure form have been successfully
analyzed by the proposed methods. Excipients and
impurities did not show interference indicating high
specificity. Results obtained were compared to those
7, 9, 11, 13
obtained by applying reference methods where
Student’s t-test and F-test were performed for comparison.
Results are shown in tables 3, 4, 5 and 6 where the
calculated t and F values were less than tabulated values for
norfloxacin, tinidazole and metronidazole which in turn
indicate that there is no significant difference between
proposed methods and reference ones relative to precision
and accuracy.
124
Table(3). Statistical analysis of results obtained by the proposed
method applied on Spara ® tablets compared with reference method.
Parameters Proposed method Reference
method (9)
N 6 6
Mean Recovery 100.49 100.58
Variance 0.864 1.450
±SD 0.929 1.640
±RSD 0.924 1.635
±SE 0.380 0.670
Student-t
0.121 (2.02)a
F-test
1.672 (5.05)b
a and b are the Theoretical Student t-values and F-ratios at p=0.05.
Table(4). Statistical analysis of results obtained by the proposed
method applied on Gatiflox ® tablets compared with reference
method.
Parameters Proposed method Reference
method (7)
N 6 6
Mean Recovery 100.42 99.64
Variance 0.595 1.520
±SD 0.771 1.806
±RSD 0.768 1.810
±SE 0.315 0.730
Student-t 0.982 (2.02)a
F-test
2.551 (5.05)b
a and b are the Theoretical Student t-values and F-ratios at p=0.05.
Table(5). Statistical analysis of results obtained by the proposed
method applied on Flagyl ® tablets compared with reference method.
Parameters Proposed method Reference
method (13)
N 6 6
Mean Recovery 99.98 100.71
Variance 0.981 1.550
±SD 0.991 1.243
±RSD 0.990 1.235
±SE 0.404 0.507
Student-t 1.130 (2.02)a
F-test
1.581 (5.05)b
a and b are the Theoretical Student t-values and F-ratios at p=0.05.
Table(6). Statistical analysis of results obtained by the proposed
method applied on Protozole ® tablets compared with reference
method.
Parameters Proposed method Reference
method (11)
N 6 6
Mean Recovery 99.55 100.65
Variance 0.637 1.445
±SD 0.798 1.331
±RSD 0.802 1.330
±SE 0.325 0.545
Student-t 1.722 (2.02)a
F-test
2.263 (5.05)b
a and b are the Theoretical Student t-values and F-ratios at p=0.05.
4. CONCLUSION:
An RP-HPLC method for rapid simultaneous estimation of
sparfloxacin, gatifloxacin, metronidazole and tinidazole
within 5 minutes was developed and validated. The
amounts obtained by the proposed method are between
99.55% and 100.49%, within the acceptance level of 95%
to 105%. The results obtained indicate that the proposed
method is rapid, accurate, selective, and reproducible.
Linearity was observed over a concentration range of 1 to
80 g.ml -1 for all four drugs. The method has been

Asian J. Pharm. Res. 2011; Vol. 1: Issue 4, Pg 119-125
[AJPRes.]
successfully applied for the analysis of marketed tablets. It
can be used for the routine analysis of formulations
containing any one of the above drugs or their combinations
without any alteration in the assay. The main advantage of
the method is the common chromatographic conditions
adopted for all formulations in addition to reduced analysis
time due to monolithic silica columns.
REFERENCES:
1. Marilyn, M., Patrick, M., Robert, W. J. vet. 172, 10–28
(2006)
2. Zechiedrich, L., Cozzarelli, R. Gen. Dev. 9, 2859–2869
(1995)
3. Blondeau, M. Clin.Ther. 21, 3-40 (1999)
4. Martindale: The Complete Drug Reference, Pharmaceutical
Press;35 edition (2007)
5. Keumhan, N., Kwang, K., Tae, J., Wonku, K. Biomed.
Chromatogr. 24(11), 1199–1202 (2010)
6. Nimagada, S., Lakshmi, N., Prabha, S., Ramesh, M. Biomed.
Chromatogr. 2(11), 1288–1295 (2008)
7. Yu, H., Zhang, L., Chen, H. Huan. Guo. Yix. Zazhi. 21(5), 52-
54 (2007)
8. Wang, C., Fu, X., Lin, L. Guan. Yao. Xue. 20(6), 623-624
(2004)
9. Cao, S., Zhang, J., Ji, X., Liu, H. Sepu. 19(5), 454-456
(2001)
10. Prachi, K., Ritu, K., Manisha, P. Int. J. Chem. Tech. Res. 2(2),
928-931 (2010)
11. Kotaiah, M., Shaik, H., Narasimha, Y., Venkateswarlu, Y.
RJPBCS. 1(4), 460-466 (2010)
12. Baing, M., Vaidya, V., Rane, J., Champanerkar, A. Anal.
Chem. 7(12), 862-865 (2008)
13. Naser, T., Jaleh, V., Farid, D., Mohammad, R. J. Pharm.
Biomed. Anal. 43(1), 325-329 (2007)
14. Minakuchi, H., Soga, N. J. Amer. Cer. Soc. 74, 2518-2530
(1992)
15. Minakuchi, H., Nakanishi, K., Soga, N., Ishizuka, N.
Chromatogr. A. 762, 135-146 (1997)
16. Phenomenex website for Onyx HPLC columns.
(http://www.phenomenex.com/phen/products/onyx/ info.html)
17. Yun-fei, Z. Dong-sheng, S. Bao-fu, Z. Chin. J. New. Drg. 9,
17 (2008)
18. An Improved Process For Gatifloxacin Preparation.
(http://www.sumobrain.com/patents/wipo/Improved-processpreparation-gatifloxacin/WO2005009970.html)
19. Kraft, M., Kochergin, P., Tsyganova, A. Pharm. Chem. J.
23(10), 861-863 (1989)
20. Guidance for Industry: Q2B of Analytical Procedures;
Methodology: International Conference of Harmonization
(ICH). Nov. (1996) (http:/www.fda.gov/eder/ guidance
/1320fnl.pdf)
125