Inayatullah Thesis Chemistry

SPECTROFLUORIMETRIC AND
SPECTROPHOTOMETRIC METHODS FOR

DETERMINATION OF FLUOROQUINOLONES IN
PHARMACEUTICAL PREPARATIONS AND
BIOLOGICAL SAMPLES
Ph. D Thesis
By
INAYATULLAH
INSTITUTE OF CHEMICAL SCIENCES

UNIVERSITY OF PESHAWAR
PAKISTAN
SEPTEMBER 2011
SPECTROFLUORIMETRIC AND
SPECTROPHOTOMETRIC METHODS FOR
DETERMINATION OF FLUOROQUINOLONES IN
PHARMACEUTICAL PREPARATIONS AND
BIOLOGICAL SAMPLES
By
INAYATULLAH
DISSERTATION
SUBMITTED TO THE UNIVERSITY OF PESHAWAR IN

PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR
THE DEGREE OF DOCTOR OF PHILOSOPHY
IN CHEMISTRY

PAKISTAN
SEPTEMBER 2011
PAKISTAN
Certified that Mr. Inayatullah S/O Hanifullah has carried out his research and
experimental work on the topic entitled as “Spectrofluorimetric and
Spectrophotometric Methods for Determination of Fluoroquinolones in
Pharmaceutical Preparations and Biological Samples” under our guidance and
supervision. His research work is original and his dissertation is worthy of presentation to
the University of Peshawar for the award of degree of Doctor of Philosophy in
Chemistry.
______________________ _______________________
SUPERVISOR CO-SUPERVISOR
Prof. Dr. Muhammad Rasul Jan Prof. Dr. Jasmin Shah
Institute of Chemical Sciences Institute of Chemical Sciences,
University of Peshawar, Pakistan University of Peshawar, Pakistan
Present address:
Vice Chancellor,
University of Malakand, Chakdara, Lower Dir,
Khyber Pakhtunkhwa, Pakistan
_____________________
EXTERNAL EXAMINER
“Every Scientific Advance is an Advance in Method. The
Invention of a New Specialized Laboratory Procedure
Brings about Rapid Conquests in New Fields of Science
and Technology. Finally, it Exhausts itself and is Replaced
by a Still More Practical Method”
To My Teacher & My Mentor
Prof. Syed Ghawas. I am indebted to him for opening my
mind and touching my heart.
To My loving wife
I thank her as the earth thanks the sun.
Your steadfastness made that which follows, real.

Acknowledgements
In the name of Allah, the Most Gracious, the Most Merciful. All praise
to Allah the Omniscient, who taught man what, he not knew.
May the peace and blessings of Allah be upon our Prophet,

Muhammad (Sallallaho alaihe wasallam), and upon his companions,
and all his true followers.
First and foremost, I am deeply indebted to my teacher and my

supervisor, Prof. Dr. Muhammad Rasul Jan for his consistent
motivation, enthusiasm for work, immense knowledge and stimulating
discussion. His constant guidance and encouragement gave me the
moral and mental support which I needed in large measure in course
of this arduous task. Only because of his support and inspiration could
I confidently embark upon a research work of such magnitude. He has
helped me improve almost every aspect of my life. I am fortunate and
proud to be his student.
Words cannot express the nature and extent of my deepest

appreciation for Prof. Dr. Jasmin Shah; my co-supervisor who is
integral to my education in the analytical sciences and inspirations to
my professional and personal development. Her involvement with her
originality has triggered and nourished my intellectual maturity that I
will benefit from, for a long time to come. Her insightful input and
encouragement are priceless during my good and bad times. I
appreciate all her contributions of time and ideas to make my Ph.D.
experience productive and stimulating. The joy and enthusiasm she
has for research work was contagious and motivational for me.
I am truly indebted to both of them. I can’t thank them enough, but I
pray Allah, to bless them for their efforts and reward them according
to His generosity.
I am really short of words to express my gratitude to my uncle Dr.

Ikramul Haq, NCE in Physical Chemistry, for his encouragement and
motivation throughout my professional and personal development.
My thanks are due to the Higher Education Commission of Pakistan,

University of Peshawar and Institute of Chemical Sciences for
providing me necessary funds, facilities and encouragement to pursue
higher education in chemistry.
Mention must be made of my worthy professors and teachers at the

Institute of Chemical Sciences who have helped me in various ways.
For this, I am truly blessed.
I give my estranged thanks to Imdadullah Muhammadzai Ph.D.,

Professor and Director Institute of Chemical Sciences and Prof. Dr.
Imtiaz Ahmad Director QEC, University of Peshawar, for the support
they gave me along the way.
Very special thanks and my deep gratitude go to Dr. Muhammad

Yaseen and Hameedullah, for helping in the literature study. Very
special thanks to Dr. Humayun Khan and Dr. Fazal Mabood, Ex-teaching
faculty of Institute of Chemical Sciences, University of Peshawar and Dr. Mir
Azam Khan, for their support in initiation of the work. I am also very
thankful to Dr. Khalid khan for providing biological samples.
I am grateful to my senior scholar colleagues for providing a

stimulating and decent environment to learn and grow. Many thanks
to Dr. Kashif Gul, Dr. Hussain Gulab, Fozia Rehman, Dr. Mumtaz Ali
and Dr. Khair Zaman. My time at the institute was made enjoyable in
large part due to my friends. I am grateful to Mr. Sultan Shah, Mr.
Atta ul Haq, Mr. Adnan, Mr. M. Naeem, Ms. Maria Sadia, Ms. Saima
Sohni, Mrs. Sadaf Durrani and Mrs. Farhat-un-Nisa Shehzad. I cannot
forget Mian Muhammad and Behisht Ara who were always around me
in my hard times.
I take this opportunity to acknowledge with thanks the help and

support that I have received from Mr. Aziz ur Rahaman (Librarian
ICS), Mr. Zaheer ud Din (store supervisor), Mr. Muhammad Ali, Mr.
Arif Ismail, Mr. Asmat Ali, Mr. Iftikhar Ahmad and all the clerical and
parateaching staff of Institute of Chemical Sciences.
Where would I be without my family? I want to express deepest

gratitude to my elderly father and mother to whom I could not give
proper time and their prayers, guidance, and sound advice have
inspired and sustained me throughout my academic and personal life.
I pay a heartfelt appreciation to my uncle Faiz-ur-rehaman, to my
brothers Muradullah, Saeedullah, Midrarullah, Ashfaqurahman,
Amjidullah and Ishtiaqurahman, to my humble and simple sisters, to
my cousin Hussain Ahmad and of course my sweet sons Abdullah
Nangyal and Adanullah Baryal and a small angel Izzah, whose love is
invaluable to me and will always sustain me. I love you all!
Inayatullah
University of Peshawar
2011
CONTENTS
S. No. TITLE Page No.
I LIST OF FIGURES i-v
II LIST OF TABLES vi-ix
III ABBREVIATIONS x-xiii
IV SUMMARY xiv-xviii
CHAPTER-1: INTRODUCTION
1.0 Introduction 1
1.1 Ciprofloxacin 15
1.1.1 Physical Characteristics 15
1.1.2 Dosage and mode of administration 16
1.1.3 Over dosage 17
1.2 Sparfloxacin 17
1.3 Levofloxacin 18

1.4 Gatifloxacin 21
1.5 Moxifloxacin 22
1.5.2 Moxifloxacin Dosing Guidelines 24
References 25
CHAPTER-2: LITERATURE REVIEW
Literature Review 38
References 75
CHAPTER-3: EXPERIMENTAL/RESULTS AND DISCUSSION
3.1.0 Investigation of spectrophotometric method for

determination of sparfloxacin in pharmaceutical preparations 88
and urine samples
3.1.1 Method development strategy for the spectrophotometric

88
determination of sparfloxacin
3.1.2 Preliminary investigation of the possibility of oxidation of 91

sparfloxacin by ammonium metavanadate for its
spectrophotometric determination
3.1.3 Optimization studies for spectrophotometric determination of

91
sparfloxacin using ammonium metavanadate
3.1.4 Verification of Beer’s law for determination of sparfloxacin by

97
the proposed spectrophotometric method
3.1.5 Application of the investigated method for the analysis of

100
sparfloxacin in various pharmaceutical brands
3.1.6 Validation of the proposed method for biological samples

104
(Artificial Urine)
3.1.7 Results and discussion 105
3.2.0 Quantification of sparfloxacin in pharmaceutical dosages and

biological samples through condensation reaction with p- 109
dimethylaminobenzaldehyde (DMAB)

109
3.2.2 Preliminary investigation of the possibility of condensation

reaction between sparfloxacin and DMAB for its 111
3.2.3 Optimization studies for spectrophotometric determination of

111
sparfloxacin using DMAB
3.2.4 Verification of Beer’s law for determination of sparfloxacin by

117
the proposed spectrophotometric method
3.2.5 Effect of common excipients found in commercial formulations

119
on determination of sparfloxacin using the proposed method
121
3.2.7 Validation of the proposed method in spiked biological samples 124
3.2.8 Determination of sparfloxacin by reference method (HPLC

125
method)
3.3.0 Indirect spectrophotometric method for determination of

132
sparfloxacin in bulk and dosage form using Ce (IV)

132
3.3.2 Preliminary investigation of the possibility of oxidation of

sparfloxacin by ammonium cerium (IV) sulphate for its 132
3.3.3 Investigation of suitable parameter for the determination of

133
sparfloxacin
3.3.4 Verification of Beer’s law and determination of limit of detection

138
and limit of quantification

141

142
3.3.7 Percent recovery of sparfloxacin from commercial formulations

143
(Standard addition method)
3.4.0 Indirect spectrofluorimetric method for determination of 148

sparfloxacin in bulk and dosage form using Ce (IV)
3.4.1 Method development strategy for the spectrofluorimetric

148
3.4.2 Preliminary investigation of the possibility of oxidation of

sparfloxacin by ammonium cerium (IV) sulphate for its 148
spectrofluorimetric determination
3.4.3 Optimization studies for spectrofluorimetric determination of

149
sparfloxacin using Ce (IV)
3.4.4 Effect of concentration of sparfloxacin on fluorescence intensity

155
of Ce (III)

157
3.5.0 Micellar-enhanced spectrofluorimetric determination of

moxifloxacin in pharmaceutical formulations and biological 165
samples

165
determination of moxifloxacin
3.5.2 Preliminary investigation of the possibility of increase in

fluorescence intensity of moxifloxacin in the presence of sodium 165
dodecyl sulphate (SDS)

167
moxifloxacin using micelle
3.5.4 Effect of concentration of moxifloxacin on fluorescence intensity 171
3.5.5 Investigation of interferences on determination of moxifloxacin 174

using the proposed method

181
moxifloxacin in various pharmaceutical brands
3.6.0 Sensitive and rapid spectrofluorimetric method for

determination of fluoroquinolones by charge-transfer 190
complexation with chloranilic acid

190
determination of fluoroquinolones
3.6.2 Preliminary investigation of the possibility of formation of

charge-transfer complex between fluoroquinolones and 191
chloranilic acid for spectrofluorimetric determination of the drugs

192
fluoroquinolones using chloranilic acid
3.6.4 Effect of concentration of fluoroquinolones on fluorescence

201
intensity of the CT complex
3.6.5 Effect of common interferers on determination of the selected

205
fluoroquinolones using the proposed method
3.6.6 Application of the investigated method for the analysis of the

213
selected drugs in various pharmaceutical brands
Conclusions 224
LIST OF FIGURES
Fig. No. Title Page No.

3.1.1 The proposed reaction mechanism of oxidation of sparfloxacin
90
by vanadate ion
3.1.2 Wavelength optimization for the spectrophotometric
94
3.1.3 Effect of concentration of ammonium metavanadate on the
95
spectrophotometric determination of sparfloxacin
3.1.4 Effect of volume of ammonium metavanadate on the
95
3.1.5 Effect of concentration of sulphuric acid on the
96
3.1.6 Effect of volume of sulphuric acid on the spectrophotometric
96
3.1.7 Effect of heating temperature on the spectrophotometric
97
3.1.8 Effect of heating time on spectrophotometric determination of
97
sparfloxacin
3.1.9 Investigation of the effect of concentration on the absorption
98
behavior of sparfloxacin
3.1.10 The effect of common excipients found in commercial
formulations on determination of sparfloxacin using the 101
proposed method
3.2.1 The proposed reaction mechanism of sparfloxacin with DMAB 110
114
3.2.3 Effect of concentration of DMAB on the spectrophotometric 114

i
3.2.4 Effect of volume of DMAB (0.3 %) on the spectrophotometric
115
115
116
3.2.7 Effect of reaction time on spectrophotometric determination of
116
sparfloxacin
118
120
3.2.10 Calibration curve for sparfloxacin using reversed phase HPLC 126
136
3.3.2 Effect of concentration of cerium (IV) on the spectrophotometric
136
3.3.3 Effect of volume of Cerium (IV) on the spectrophotometric
137
137
138
3.3.6 Effect of heating time on spectrophotometric determination of
138
sparfloxacin
3.3.7 Effect of concentration on the absorption behavior of
139
sparfloxacin
3.3.8 Effect of common excipients on determination of sparfloxacin 142
3.4.1 Excitation (1) and emission (2) spectra of cerium (III) generated 152
ii
after reaction with sparfloxacin
3.4.2 Effect of concentration of ammonium cerium (IV) sulphate on
153
the spectrofluorimetric determination of sparfloxacin
3.4.3 Effect of volume of ammonium cerium (IV) sulphate on the
153
spectrofluorimetric determination of sparfloxacin
154
3.4.5 Effect of heating temperature on the spectrofluorimetric
154
3.4.6 Effect of heating time on spectrofluorimetric determination of
155
sparfloxacin
3.4.7 Stability of fluorescence intenisty of Ce (III) 155
3.4.8 Effect of concentration of the drug on the fluorimetric behavior
156
of Ce (III)
3.4.9 Interferences effect of common excipients found in commercial
proposed method
3.5.1 Fluorescence intensity of the analyte as a function of medium. 166
3.5.2 Effect of pH on the fluorescence intensity of moxifloxacin 166
3.5.3 Excitation and emission spectra of moxifloxacin with and
169
without SDS
3.5.4 Effect of concentration of SDS on the spectrofluorimetric
170
3.5.5 Effect of volume of SDS (0.15 %) on the spectrofluorimetric
170
3.5.6 Effect of temperature on the fluorescence intensity of
171
moxifloxacin
3.5.7 Effect of concentration on fluorimetric behaviour of
172
moxifloxacin
3.5.8 Calibration curve for spectrofluorimetric determination of 172
iii
moxifloxacin at lower concentration level
176
on determination of moxifloxacin using the proposed method
3.5.10 Effect of common cations found in human plasma and urine
samples on determination of moxifloxacin using the proposed 178
method
3.5.11 Effect of common compounds occurring in human urine samples
179
on determination of moxifloxacin (0.1 µg/mL)
3.5.12 Effect of co-administered drugs on determination of
181
moxifloxacin using the proposed method (0.1 µg/mL)
3.6.1 The proposed reaction mechanism of fluoroquinolones with CL
191
acid
3.6.2 Excitation and emission spectra of levofloxacin only ( ) and
195
CT complex (-----) with chloranilic acid
3.6.3 Excitation and emission spectra of moxifloxacin only ( ) and
196
3.6.4 Excitation and emission spectra of ciprofloxacin only ( ) and
196
3.6.5 Excitation and emission spectra of gatifloxacin only ( ) and
197
3.6.6 Fluorescence intensity of the CT complex as function of the
197
medium
3.6.7 Effect of concentration of CL acid on the spectrofluorimetric
198
determination of levofloxacin
198
199
determination of ciprofloxacin
199
determination of gatifloxacin
3.6.11 Effect of volume of CL acid on the spectrofluorimetric 200
iv
determination of the selected fluoroquinolones
3.6.12 Effect of temperature on fluorescence intenisty of the CT
200
complex
3.6.13 Stability of fluorescence intenisty of the CT complex 201
3.6.14 Investigation of the effect of concentration of levofloxacin on
202
the fluorimetric behavior of CT complex
3.6.15 Investigation of the effect of concentration of moxifloxacin on
202
3.6.16 Investigation of the effect of concentration of ciprofloxacin on
203
3.6.17 Investigation of the effect of concentration of gatifloxacin on the
203
fluorimetric behavior of CT complex
3.6.18 Interferences effect of common excipients on % recovery of the
207
analyte (1.0 µg/mL) using the proposed method
samples on determination of the selected fluoroquinolones (1.0 209
µg/mL)
3.6.20 Effect of common compounds occurring in human urine samples
210
on determination of levofloxacin (0.5 µg/mL)
3.6.21 Effect of co-administered drugs on determination the selected
213
FQ using the proposed method (0.5 µg/mL)
v
LIST OF TABLES
Table No.
Title Page No.
1.1 Pharmacokinetic parameters of fluoroquinolones 10
98
3.1.2 Analytical parameters for the spectrophotometric
100
3.1.3 The effect of common excipients found in commercial
proposed method
3.1.4 Evaluation of accuracy and precision of the proposed method
102
for sparfloxacin determination
3.1.5 Evaluation of percent recovery of sparfloxacin from
103
commercial formulations by the proposed method
3.1.6 Determination of active ingredients in the commercial form 104
3.1.7 Composition of artificial urine medium 105
3.1.8 Evaluation of percent recovery of sparfloxacin from artificial
105
urine medium by the proposed method
3.2.1 Effect of the order of mixing of the reagents on absorbance
116
117
119
3.2.4 Effect of common excipients on % recovery of the analyte
120
for determination of sparfloxacin 121
3.2.6 Evaluation of percent recovery of sparfloxacin from 122

vi
3.2.7 Determination of active ingredients in the commercial
123
formulations
3.2.8 Spectrophotometric determination of sparfloxacin in spiked
125
urine by the proposed method
3.2.9 Spectrophotometric determination of sparfloxacin in spiked
125
plasma by the proposed method
3.2.10 Determination of sparfloxacin in commercial formulation
127
and validation by reference method
3.3.1 Effect of concentration on the absorption behavior of
139
sparfloxacin
140
3.3.3 Effect of common excipients on determination of
141
sparfloxacin
143
formulations
143
3.3.6 Evaluation of percent recovery of sparfloxacin from
144
3.4.1 Effect of concentration of the drug on the fluorimetric
156
behavior of Ce (III)
3.4.2 Analytical parameters for the spectrofluorimetric
157
3.4.3 Interferences effect of common excipients found in
commercial formulations on determination of sparfloxacin 158
159
3.4.5 Evaluation of recovery (%) of sparfloxacin from commercial 160
vii
formulations by the proposed method
161
formulations
3.5.1 Effect of concentration on fluorimetric behaviour of
172
moxifloxacin
3.5.2 Analytical characteristics for the spectrofluorimetric
173
3.5.3 Effect of common excipients found in commercial
formulations on determination of moxifloxacin (0.1 µg/mL) 175
samples on determination of moxifloxacin using the 177
proposed method
3.5.5 Effect of common compounds occurring in human urine
179
samples on determination of moxifloxacin (0.1 µg/mL)
3.5.6 Effect of co-administered drugs on determination of
180
moxifloxacin using the proposed method (0.1 µg/mL)
182
for moxifloxacin determination (n = 3)
3.5.8 Evaluation of percent recovery of moxifloxacin from
183
184
formulations
3.5.10 Spectrofluorimetric determination of moxifloxacin in spiked
185
3.5.11 Spectrofluorimetric determination of moxifloxacin in spiked
185
3.6.1 Optical parameters for the spectrofluorimetric determination
204
of the selected fluoroquinolones by the proposed method
3.6.2 Interferences effect of common excipients on % recovery of 206
viii
the analyte (1.0 µg/mL) using the proposed method
samples on determination of the selected fluoroquinolones 208
(1.0 µg/mL)
3.6.4 Effect of common compounds occurring in human urine
210
samples on determination of levofloxacin (0.5 µg/mL)
3.6.5 Effect of co-administered drugs on determination of the
212
selected FQ using the proposed method (0.5 µg/mL)
214
for the determination of selected fluoroquinolones
3.6.7 Evaluation of percent recovery of the selected drugs from
216
218
formulations
3.6.9 Spectrofluorimetric determination of levofloxacin in spiked
219
3.6.10 Spectrofluorimetric determination of levofloxacin in spiked
220
ABBREVIATIONS
ix
% Percent
°C Degree celsius
µg Microgram
µg/mL Microgram per milliliter
µm Micrometer
AM1 Austin Model 1
AUC Area under the concentration–time curve
BCG Bromocresol green
BPB Bromophenol blue
Cipro Ciprofloxacin
CL Chloranilic acid
cm Centimeter
Cmax Maximum concentration
CSF Cerebrospinal fluid
CT Charge transfer
CZE Capillary zone electrophoresis
DMAB p-dimethylaminobenzaldehyde
DNA Deoxyribonucleic Acid
Ɛ Molar absorptivity
E. dps Eye drops
ECG Electricocardiography
F Bioavailability
FDA Food and Drug Administration
FQs Fluoroquinolones
g Gram
x
g/cm3 Gram per centimeter cube
g/mol Gram per mole
Gati Gatifloxacin
HPLC High performance liquid chromatography
ICH International conference on harmonization
Inf. Infusion
KJ/mol Kilojoule per mole
L Liter
LC Liquid chromatography
LC–ESI-MS/MS Liquid chromatography- electron spray ionization

mass spectrometry
Lev Levofloxacin
LEV-BCG Levofloxacin-Bromocresol Green
LEV-BPB Levofloxacin-Bromophenol Blue
ln Natural logarithm
LOD Limit of detection
log Common logarithm
LOQ Limit of quantification
Ltd Limited
M Molarity
mg Milligram
mg/g Milligram per gram
mg/L Milligram per liter
mg/tab Milligram per tablet
MIC Minimum Inhibitory Concentration
xi
min Minute
mL Milliliter
mM millimolar
mmol/L milli mole per liter
mol Mole
mol/L Mole per liter
mol/L/cm Mole per liter per centimeter
Moxi Moxifloxacin
MS Mass spectrometry
N Normal
nm Nanometer
Pvt Private
QC Quality control
r2 Correlation coefficient
RP Reversed Phase
RP-HPLC Reversed Phase High Performance Liquid

Chromatography
RSD Relative standard deviation
S Standard deviation
SDS Sodium dodecyl sulfate
SIM Selective Ion monitoring
SPE Solid phase extraction
SPME–GC–MS Solid-phase microextraction–gas chromatography–

mass spectrometry
SPMTE Solid phase membrane tip extraction
SPR Surface plasmon resonance
xii
SRM selected reaction monitoring
SWV Square wave voltammetry
T Time
T1/2 half-life
Tab. Tablet
TLC Thin layer chromatography
Tmax Time to achieve maximal concentration
UV Ultra violet
V Volt
Vd Volume of distribution
Wt Weight
xiii
Summary
SUMMARY
Chemical assay of a drug in its bulk, pharmaceutical preparations and biological samples
is extremely important from the health point of view and this requires analytical methods
possessing the required sensitivity, selectivity, accuracy and reproducibility for
determination of active ingredients of the drugs. Most of the reported methods for
determination of fluoroquinolones antibiotics not only require well equipped laboratories
with sophisticated and expensive instruments, solvents but also highly skilled manpower
to run these equipments. Keeping in mind the technical and financial constraints, it is
imperative to explore new methods based on the use of simple and inexpensive
instruments like spectrophotometer and spectrofluorometer while maintaining the same
level of accuracy. The present work is an attempt in the same direction.
Aim of the present research work was to investigate and develop fast, inexpensive and
relatively simpler spectrofluorimetric and spectrophotometric methods for determination
of fluoroquinolones widely prescribed and manufactured by the local units. The methods
developed will enable the local pharmaceutical industries and officials of public health
departments to adopt them as standard quality control protocols on the basis of their
simplicity, accuracy and non-involvement of sophisticated and expensive instruments.
The first chapter of the thesis is concerned with introduction of fluoroquinolones. It

includes classification, pharmacology and pharmacokinetics of fluoroquinolones and
information regarding physical and chemical aspects of fluoroquinolones (ciprofloxacin,
levofloxacin, sparfloxacin, gatifloxacin and moxifloxacin) selected for this work.
The second chapter covers a thorough review of the relevant literature reported for
monitoring of these drugs. This chapter includes chromatographic, spectrophotometric,
spectrofluorimetric, voltammetric and other analytical techniques used for determination
of ciprofloxacin, levofloxacin, sparfloxacin, gatifloxacin and moxifloxacin in various
commercial formulations and body fluids. The methods have been discussed with
emphasis on scope, type of sample, reagents used, linearity range and limits of detection
and quantification.
xiv
Summary
The third and last chapter contains experimental work with the results and discussion of
various methods investigated for the analysis of active ingredients in various commercial
formulations and validation in spiked biological samples. This chapter is sub divided into
two sections. The first section describes spectrophotometric methods for determination of
sparfloxacin and second section is concerned with spectrofluorimetric methods for
determination of ciprofloxacin, levofloxacin, sparfloxacin, gatifloxacin and moxifloxacin.
Each section is further sub divided into three parts, each covering methods development
for determination of selected fluoroquinolones.
Part I of this chapter deals with method development strategy and the proposed chemical
reactions. Sparfloxacin was oxidized into red coloured product using ammonium
monovanadate in acidic media. At optimum conditions the coloured product showed
maximum absorption at 530 nm with molar absorptivity of 4.0 x 10 3 L/mol/cm, and
linearity in the concentration range 0.8-28.0 µg/mL of sparfloxacin. The proposed
method was successfully applied for determination of sparfloxacin in different
commercial pharmaceutical preparations (tablets) and synthetic urine sample spiked with
the sparfloxacin. The influence of commonly used excipients on the determination of
sparfloxacin was studied. Percentage recoveries in the range of 98.0 % ± 0.14 – 100.0 %
± 0.20 were obtained. The observed data has been evaluated statistically which showed
high accuracy and precision.
Part II of this chapter deals with determination of sparfloxacin through condensation

reaction with p-dimethylaminobenzaldehyde (DMAB). Sparfloxacin is reacted with
DMAB in acidic medium at room temperature to produce a yellow coloured condensation
product, having λmax at 392 nm with molar absorptivity of 4.9 x 10 3 L/mol/cm. All
variables affecting the reaction, like reagent concentration, molarity of sulphuric acid and
reaction temperature were carefully studied and optimized. Under the optimum
conditions, linear relationship with good correlation coefficient (0.9997) was found
between absorbance of the product and the concentration of sparfloxacin in the range of
2.0 – 80.0 µg/mL. The limits of detection and quantification were 0.22 and 0.75 µg/mL
respectively. The reliability of the method was checked at three different concentrations
of the sparfloxacin and was found satisfactory; the value of relative standard deviation
xv
Summary
did not exceed 3.8 %. The method was found free of the interferences effect of the
excipients commonly present in dosage forms. The proposed method was statistically
validated and applied to the analysis of the drug in pure form, pharmaceutical dosage
forms, and spiked biological fluids with good accuracy and precision. The percentage
recovery ranged from 99.0 – 100 ± 1.3 %. The results obtained by the proposed method
were compared with those obtained by the literature HPLC method.
Part III of this chapter deals with spectrophotometric method for determination of
sparfloxacin in bulk and dosage form using Ce (IV). The proposed method is based on
oxidation of sparfloxacin by cerium (IV) in acidic medium with subsequent measurement
of decrease in absorbance of cerium (IV) at 315 nm. Different variables affecting the
reaction such as concentration and volume of cerium (IV), type and concentration of
acidic medium, heating temperature and heating time were carefully optimized. Under
the optimum conditions, Beer’s law was obeyed in the concentration range of 0.02 – 0.2
µg/mL. No interferences were observed from the common formulations excipients
present in the dosage form of the drug. The proposed method was successfully applied to
the analysis of the investigated drug in pure and pharmaceutical formulations with good
accuracy and precision. The percentage recovery ranged from 95.0 ± 3.7 - 102.5 ± 1.7 %.
Part IV of this chapter deals with spectrofluorimetric determination of sparfloxacin in

bulk and dosage form using Ce (IV). The proposed method is based on oxidation of
sparfloxacin by cerium (IV) in acidic medium followed by measurement of fluorescence
intensity of the product cerium (III) at 352 nm with excitation wavelength 250 nm.
Different variables affecting the reaction such as concentration and volume of cerium
(IV), type and concentration of acidic medium, heating temperature and heating time
were carefully studied and optimized. Under the optimum conditions, linear range of the
method was found to be 0.02 – 0.1 µg/mL. No interferences were observed from the
common formulations excipients present in the dosage form of the drug. The proposed
method was successfully applied to the analysis of the investigated drug in pure and
pharmaceutical formulations with good accuracy and precision. The percentage recovery
ranged from 93.0 ± 2.7 % – 102.2 ± 2.3 %.
xvi
Summary
Part V of this chapter is concerned with micellar-enhanced spectrofluorometric

determination of moxifloxacin in pharmaceutical formulations and biological samples.
Simple, rapid, specific and highly sensitive spectrofluorometric method has been
developed for the quantification of moxifloxacin in pure form, commercial formulations,
human urine and plasma using sodium dodecyl sulphate surfactant micelle. Different
experimental parameters that effect fluorescence intensity of the drug were studied and
optimized. A linear relationship was found between fluorescence intensity and
moxifloxacin concentration in the range of 1.25 ng/mL to 3200 ng/mL. The detection and
quantification limits were found to be 0.186 and 0.62 ng/mL respectively. The proposed
method was applied for quantification of moxifloxacin in tablets, infusion and eye drops
with mean percentage recovery of 99.89 %. Common excipients used as additives in
pharmaceutical formulations do not interfere with the method. The method was
successfully applied for quantification of moxifloxacin in spiked human urine and
plasma. The effect of some common cations and compounds present in urine and plasma
were also investigated and the method was found free of interferences. The effect of co-
administered analgesic and other drugs were also studied and no interferences were
found.
Part VI of the 3rd chapter deals with sensitive and rapid spectrofluorimetric method for
determination of selected fluoroquinolones with chloranilic (CL) acid. The method is
based on the monitoring of fluorescence intensity of the charged-transfer (CT) complex
formed between chloranilic acid and ciprofloxacin, levofloxacin, gatifloxacin and
moxifloxacin. The CT complex is stable and has a distinct fluorescence activity than the
respective drugs. Experimental parameters that effect fluorescence intensity of the
complex were carefully optimized. The method allows determination of ciprofloxacin,
levofloxacin, gatifloxacin and moxifloxacin in the concentration range of 0.02 – 10.0
µg/mL, 0.06 – 3.20 µg/mL, 0.02 – 10.0 µg/mL and 0.02 – 8.0 µg/mL respectively with
good correlation coefficient in the range of 0.9929 – 1.0 in methanolic medium. The
assay limits of detection and quantification were 0.005 µg/mL and 0.018 µg/mL, 0.012
µg/mL and 0.04 µg/mL, 0.006 µg/mL and 0.019 µg/mL and 0.008 µg/mL and 0.019
µg/mL for ciprofloxacin, levofloxacin, gatifloxacin and moxifloxacin respectively. The
method was applied for determination of the drugs in tablets, infusion and eye drops
xvii
Summary
formulations with mean percentage recovery of 101.58 % with high accuracy. Common
excipients used as additive in pharmaceutical preparations, some common cations and
compounds present in urine and plasma, and co-administered analgesic, vitamins and
other drugs do not interfere with the method. The method was successfully applied for
the quantification of the selected fluoroquinolones in spiked human urine and plasma
samples.
xviii
INTRODUCTION
Introduction
1.0 Introduction[1-45]
A Pharmaceutical drug is defined as any chemical substance prescribed for the

treatment, cure, and prevention of disease or medical diagnosis.
Classification
Pharmaceutical drugs have been classified in a number of ways. A sample of classes of

medicine includes:
1. Antipyretics: the drugs intended for reducing fever effects.

2. Analgesics: the drugs used for relieving pain.
3. Antimalarial drugs: the drugs intended for treating malaria.
4. Antiseptic: the drugs intended for preventing germ growth near injuries, cuts,
gashes and wounds.
5. Antibiotic: the drugs intended for inhibiting germ activities.
Defining antibiotics
The word “antibiotics” is derived from Greek language and is made up of two parts
“anti” means against and “bios” means life. Antibiotics are chemicals produced by or
derived from microorganisms in one way or the other. Antibiotics are drugs that either
destroy bacteria or prevent their reproduction. Antibiotics that kill bacteria are called
“bactericidal” and the ones that stop the growth of bacteria are called “bacteriostatic”.
The first antibiotic “penicillin” was discovered by Alexander Fleming in 1928 and was a
significant breakthrough for medical science.
Penicillin was introduced to the market during the 1940s and since then scientists are
busy to discover and develop other antibiotics and there are a number of success stories.
Today, there are over 100 different types of antibiotics at human disposal. About 90% of
antibiotics are obtained from living organisms such as bacteria; others are prepared
synthetically, either in whole or in part.
1
Introduction
Classification of antibiotics
Although antibiotics can be classified by a number of schemes, based on antibacterial

spectrum (narrow or broad) or route of administration (oral, parenteral, or topical), or
type of activity (bacteriostatic or bactericidal), the very common and useful one is based
on chemical structure. Structurally related antibiotics will generally have similar mode of
action, effectiveness, adverse effects and allergic potential.
The commonly used classes of antibiotics are: Quinolones, fluoroquinolones, penicillins,

cephalosporins, macrolides, and tetracyclines. Each class of antibiotics is further
composed of a number of generations and each generation contains multiple drugs and
each drug is unique in some way.
Quinolones
Quinolones are excellent antibiotics that have proven to be helpful in a number of clinical
indications. In the years following their launch, they became extremely popular, and their
consumption increased rapidly, both in human medicine and in animal food preparation.
Quinolone antibacterial research and development has enjoyed an enormous worldwide

effort since its discovery. The very first member of the family, nalidixic acid was
accidentally discovered in early 1960s by Lescher and his colleagues as a byproduct
during their work on synthesis of antimalarial compound chloroquine. During this time,
more than 10,000 structurally related agents have been described in hundreds of patents
and journal articles. The product of this wealth of research has been a continually
improving progression of marketed quinolone antibacterial agents. Form the early days of
Gram-negative selective agents, limited to treatment of urinary tract infection, the field
has matured to provide broad spectrum drugs capable of treating not only urinary tract
infections but also systemic infections caused by Gram-positive and Gram-negative
pathogens at sites ranging from skin to joints to the respiratory tract. Some newer agents
incorporate activity against anaerobic pathogens, making them useful in surgical and
gynecological infections. The pharmacokinetic performance of these agents has also been
optimized, allowing for once-daily dosing of a subset of the newer fluoroquinolones.
2
Introduction
Fluoroquinolones
Fluoroquinolones is a family of one of the most successful and important classes of

manmade antibiotics which has a very broad spectrum of activity against Gram-positive
and Gram-negative pathogens. Their generic name often contains the suffix “floxacin”.
This group of antimicrobial agents has a long history of use in clinical practice. First it
was used to treat the infections caused by Gram-negative bacteria only and more recently
new derivatives have been developed with increased activities against Gram positive
bacteria such as Streptococcus pneumonia, the most common bacteria causing infections
in soft tissues, acute otitis and meningitis.
The 4-quinolones, which were synthesized within a decade after the discovery of
nalidixic acid, had its spectrum of activity against a limited range of Gram-negative
bacteria. Parallel developments in Japan had yielded 7-piperazine substituted compounds,
which had limited activity against Pseudomonas aeruginosa. However, the breakthrough
to broad spectrum activity waited a further 10 years before fluorination, primarily at the
6- position, resulted in fluoroquinolones. These structural changes in the parent molecule
yielded agents that were prescribed for a number of indications, including gastrointestinal
tracts, genitourinary, respiratory, skin and soft tissues and other structural infections. In
most bodily tissues and fluids, the fluoroquinolones are characterized by excellent
penetration and therapeutic ratios. Since mid-1980s, fluoroquinolones have become a key
group of manmade antibiotics with activity that covers a broad range of Gram-positive
and Gram-negative pathogens.
A number of new fluoroquinolones were introduced in the following years and were
withdrawn due to their rare but severe adverse effects. Fortunately, the 8-
methoxyquinolones, moxifloxacin and gatifloxacin, which are highly potent against S-
pneumoniae (10 fold greater than the earlier second generation agents), were found
clinically effective and appear free from either significant or unexpected toxicity, filled
this therapeutic vacuum. Their proven activity against S. pneumonia, coupled with
maintained high potency against Haemophilus influenza and Moraxella catarrhalis, and
excellent penetration into respiratory tissues, including the intracellular habitat of
3
Introduction
Chlamydia and Legionella of Chlamydia and Legionella spp., suggests that, where
ciprofloxacin was considered by many to be inappropriate for respiratory infections, 8-
methoxyquinolone derivatives will now become agents of choice.
Fluoroquinolones are receiving a significant attention due to their broad spectrum of

activity, potency, efficacy, variable indications, well oral bioavailability, excellent tissue
penetration, once daily dosing, longer elimination half-life and excellent pharmacokinetic
profile. As a result, they are known a therapeutically effective class of antibacterial
agents over the last decade. No other classes of antimicrobial agents have progressed so
rapidly or have been advanced with such zeal by pharmaceutical research organizations.
This class is among the world’s most used antimicrobial drugs in community and hospital
settings.
The fluoroquinolones and their precursors have a number of predictable structure–

activity and structure–adverse effect relationships relating to nuclear and side chain
configurations. Thus, the design of new molecules can avoid many of the problems that
have been characterized with previous members of the group. It may be anticipated that
further modifications to the molecular structure will improve spectrum and activity while
reducing the incidence of adverse effects.
Ciprofloxacin, moxifloxacin, levofloxacin, norfloxacin, sparfloxacin, ofloxacin,

lomefloxacin are some of the commonly used fluoroquinolones.
Classification of fluoroquinolones
No standard classification of quinolones antibiotics into generation is available in the

literature, however, nalidixic acid, which was introduced in the pharmaceutical market in
1962, is considered as the first generation quinolone. Since then numerous structural
modifications in the parent quinoline nucleus to improve antimicrobial activity and
advance pharmacokinetics performance, has given birth to a number of derivatives,
which have been divided into generations. The fluoroquinolones are classified into
generations based on their antibacterial spectrum, with earlier generation quinolones
4
Introduction
more effective against Gram-negative bacteria and the spectrum of activity expanding to
include more Gram-positive bacteria with the later generations.
I. First generation
Nalidixic acid, Cinoxacin, flumequine and pipemidic acid are the first-generation
members, which are the oldest and the least used quinolones. Systemic distribution of
these drugs was poor. They had limited activity and were used primarily for urinary tract
infections caused by Gram-negative pathogen. They were effective against Gram-
negative bacteria but they lacked activity against Gram-positive pathogens and
anaerobes. The first generation quinolones required multiple dosing and they were more
prone to the development of bacterial resistance.
II. Second generation
The late 1970s and early 1980s was the era of second generation of quinolones,
particularly fluoroquinolones. The activities of the second-generation fluoroquinolones
against Gram-negative has been broadened and some Gram-positive and atypical
pathogen have been covered. In addition, drugs belonging to this generation showed
better pharmacokinetics with the introduction of piperdinyl substituent. In contrast to
first-generation quinolones, these drugs have more success stories in the treatment of
pyelonephritis, skin infections, sexually transmitted diseases, complicated urinary tract
infections and selected pneumonias.
Ciprofloxacin is the most common and widely used second-generation fluoroquinolone.

It is the most effective fluoroquinolone against Pseudomonas aeruginosa. The next most
commonly used second generation fluoroquinolone is ofloxacin. These are available in
oral and parenteral formulations. Some other common agents include enoxacin,
lomefloxacin, norfloxacin and enrofloxacin.
5
Introduction
III. Third generation
This class of fluoroquinolones was introduced to mark the expanded activity against
Gram-positive pathogens. This class is effective particularly against penicillin-sensitive
and penicillin-resistant Streptococcus pneumonia, and atypical pathogens such as
Mycoplasma pneumoniae and Chlamydia pneumoniae. Although the third-generation
fluoroquinolones retain activity against Gram-negative, they are less potent than
ciprofloxacin against Pseudomonas species.
Because of their broad antibacterial spectrum, third-generation fluoroquinolones are

beneficial in the treatment of acute exacerbations of chronic bronchitis, community-
acquired pneumonia, acute sinusitis which are their main target indications. Levofloxacin
is the most well-known and widely used third-generation fluoroquinolone. Other
members of the generation are gatifloxacin and sparfloxacin.
IV. Fourth generation
The fourth generation fluoroquinolone has maintained activities against Gram-negative

and has higher efficacy against Gram-positive bacteria and have extended its spectrum of
activity to anaerobes. They also maintain activity against Pseudomonas species which is
comparable to that of second generation fluoroquinolones such as ciprofloxacin.
Trovafloxacin and moxifloxacin are its very common examples.
Mode of action
Fluoroquinolones are transported across the bacterial cell membrane into bacterial
cytoplasm by transport protein. Classically, DNA gyrase was considered the enzymatic
target of the quinolone antibacterials. Topoisomerase IV, an enzyme discovered in 1990,
is now recognized as another important biochemical target in bacteria for
fluoroquinolones, particularly in Gram-positive pathogens. A clearer understanding of the
roles played by topoisomerase IV and DNA gyrase in replication of the bacterial
chromosome has helped to expound mechanism of bactericidal action of quinolones.
6
Introduction
Replication of DNA
When considering the mechanism by which quinolones kill bacteria, much attention has
been focused on the ability of these agents to inhibit the catalytic activity of DNA gyrase.
However, for complete understanding of their mechanism of action, the implications of
this enzyme inhibition on replication of the bacterial chromosome need to be considered.
Inside the bacterium, the circular chromosome has to be folded inside a cell that is 1 – 2
μm long. Relaxed DNA exists as a helical molecule. Topoisomerases are crucial enzymes
which maintain cellular DNA in the appropriate state of supercoiling in both replicating
and non-replicating regions of the chromosome. The process of strand separation at the
replication site drastically alters the degree of supercoiling of DNA and topoisomerases
are key enzymes that adjust this shift in supercoiling both ahead of and behind the
replication fork.
DNA replication in bacteria is a highly regulated process and is strictly coordinated with
the growth rate of the cell. It begins at a specific sequence on the chromosome and
proceeds along the length of the chromosome in a bidirectional manner. DNA helicase
break the hydrogen bonds between the bases in two DNA strands, unwinds the duplex
DNA substrate ahead of DNA polymerase and stabilizes the exposed single strand
preventing them from joining back together. The points at which the two strand of DNA
separates to allow replication of DNA are known as replication forks. The replication
forks continue around the circular chromosome until they meet at the opposite end of the
molecule or encounter replication termination sequences. The enzyme DNA polymerase
then moves along each strand of DNA behind each replication fork, synthesizing new
DNA strand exactly similar to the original one. The combined action of helicase and
DNA polymerase introduces positive super helical twist ahead of the replication fork. If
left unresolved, these positive super helical twists would eventually stop progression of
the replication fork. The bacterial enzyme, DNA gyrase, which is also known as
topoisomerase II, is responsible for removing the positive super helical twists in the DNA
structure. DNA gyrase works ahead of the replication fork to remove excess positive
super helical twists, so that DNA replication could proceed. With the combined
7
Introduction
involvement of this enzyme, an entire duplicate copy of the bacterial genome is produced
as the two replication forces move in opposite direction around the entire DNA genome.
Eventually, as the two replication forces meet, two new complete chromosomes have
been made, each consisting of one old and one new strand of DNA. In order to allow the
two new interlinked chromosomes to go apart, another bacterial enzyme is needed which
is known as topoisomerase IV. This enzyme is structurally related to DNA gyrase.
Topoisomerase IV conducts separation of the linked daughter DNA molecules after
replication is complete, so that they can be segregated into two new daughter bacterial
cells.
Quinolone antibiotics are synthetic molecules that are bactericidal. The potency of these
drugs is greatly improved by the addition of fluorine moiety at the position 6 and thus
these drugs are termed as fluoroquinolones. The fluoroquinolones rapidly inhibit bacterial
DNA synthesis, resulting in bacterial cell death. Fluoroquinolones act by inhibiting the
activity of both the DNA gyrase and topoisomerase IV enzymes. For most Gram–
negative bacteria, DNA gyrase is the primary fluoroquinolones target. It has been shown
that fluoroquinolones bind specifically to the complex of DNA and DNA gyrase, rather
than the DNA or DNA gyrase alone. Consequently quinolone stabilizes the enzyme-DNA
complexes, which in turn results in breaks in the DNA that are fatal to the bacterium.
A second mechanism of fluoroquinolones action, with some exception, is interaction with

another enzyme topoisomerase IV. This enzyme is the primary target of fluoroquinolones
in most Gram positive bacteria, with DNA gyrase being the secondary target. The
separation of two new interlinked daughter strands of the DNA is disrupted. The final
result on the bacteria, however, is the same. Bacterial replication is disrupted and the
bacterium breaks apart.
The relative potency of different fluoroquinolones antibiotics, and thus their spectrum of
activity are dependent in part on their affinity for either DNA gyrase or topoisomerase IV
or both.
8
Introduction
Pharmacokinetics [46-87]
The ultimate goal for the usage of fluoroquinolones is to eradicate bacterial pathogens at
specific sites of infection, eventually leading to clinical success in the human body
without causing adverse reactions. In order to accomplish this, an understanding of
pharmacokinetic and pharmacodynamics concepts is crucial.
The pharmacokinetics of antibiotics is the study of antibiotic transit from administration

site to specific infection site, and eventually to elimination. This process consists of three
steps: absorption, distribution, and elimination. Pharmacokinetic properties are pivotal
factors for selecting a judicious antibiotic and choosing an appropriate dosing regimen in
order to maximize bacterial eradication and to minimize resistance. Since an antibiotic
exerts sufficient bactericidal activity only when it reaches and stays at infection sites,
clinicians should be familiar not only with the drug’s antibacterial activity, but also with
its bioavailability, serum concentrations, tissue penetration, mechanism of metabolism
and elimination, drug and food interactions, drug-and-drug interactions, drug-and-disease
interaction, and other factors that can alter its pharmacokinetics. Moreover, with an
understanding that various patient populations can influence half-life or clearance
differently, clinicians will be able to select proper doses and dosing schedules needed to
optimize antibiotic activity while keeping avoidable side effects and costs to a minimum.
Absorption
Absorption refers to the passage of drug from the drug’s administration site in the body to
the general circulation. Bioavailability (F), maximum concentration (C max), and time to
achieve maximal concentration (Tmax) are useful parameters that describe drug absorption.
Bioavailability
The bioavailability (F) of a drug refers to the fraction of the administered dose that is
absorbed into the bloodstream. Theoretically speaking, bioavailability is 100% after
intravenous administration because the entire dose is infused into the systemic
9
Introduction
circulation. However, following oral administration of a drug, bioavailability ranges from

0 % (no drug absorption) to 100 % (complete drug absorption).
As shown in Table 1.1, oral formulations of fluoroquinolones exhibit excellent

bioavailability. For example, the bioavailability of ciprofloxacin has been reported to be
70–85 %. Even more impressive bioavailabilities have been observed with other
fluoroquinolones, including levofloxacin, moxifloxacin, sparfloxacin, and gatifloxacin.
Their oral administrated dose is comparable to their intravenous dose. This outstanding
feature enables these fluoroquinolones to target a market for treatment of community-
acquired pneumonia since they also have well to excellent antimicrobial activity against
most typical respiratory tract pathogens, including penicillin-resistant Streptococcus
pneumoniae.
Table 1.1 Pharmacokinetic parameters of fluoroquinolones
Drug Dose Cmax Tmax Bioavaila Vd Protein T1/2 AUC

(mg) (µg/mL) (hr) bility (%) (l/Kg) binding (hr) (µg.hr/
(%) mL
Ciprofloxacin 500 2.4 1.2 70 – 85 2.1– 2.5 35 3–5 11.6
Levofloxacin 500 5.5 – 6.2 1–2 99 1.09 24 – 38 7–8 47.7
Sparfloxacin 100 0.78 – 0.98 5.33 92 ------- 37 - 45 14 - 16 18 – 23
Gatifloxacin 400 3.4 – 3.8 1–2 96 – 98 1.5 – 2.0 20 7–8 32.4
Moxifloxacin 400 3.1 – 4.5 1-2 86 – 92 1.84 30 - 50 12 - 13 31 - 48
Cmax = maximum drug concentration; T max = time to reach peak concentration; V d =

volume of distribution, T1/2 = half-life, AUC = area under the concentration–time curve.
Distribution
I. Volume of distribution
The volume of distribution (Vd) of a drug is defined as the theoretical body space
available for its distribution. It is calculated by dividing the amount of drug administered
by the peak plasma concentration. High values of V d (>1 liter/kg) indicate extravascular
10
Introduction
penetration. As listed in Table 1.1, the V d values of fluoroquinolones are above 1 liter/kg,
suggesting extensive distribution into tissue compartments. Fluoroquinolones are not
highly protein bound. As shown in Table 1.1, protein binding of commonly used
fluoroquinolones ranges from about 20 % for gatifloxacin to 70 % for trovafloxacin.
Relatively low protein binding may contribute to the high V d observed in
fluoroquinolones.
II. Extravascular penetration fluid, cellular and tissue penetration
Fluoroquinolones achieve sufficient penetration into body fluids and tissues through
passive diffusion across the capillary bed. In case of kidney penetration, active transport
mechanisms also play a role. High extravascular penetration has been shown for all the
fluoroquinolones. In general, fluoroquinolones have been documented to penetrate well
into various fluids and tissues, including bile, pancreatic juice, peritoneal fluid, pleural
exudate, and empyema, aqueous humor, semen, human milk, joint fluid, liver, colon,
bone tissue, muscle, fat, skin, and pharyngeal mucosa, cartilage, gall bladder wall,
muscle, and skin. Even though the extent of penetration is different depending on the
agent and tissue, favorable tissue penetration is a common feature of fluoroquinolones.
III. Drug penetration into respiratory tract system
Fluoroquinolones achieve good penetration into respiratory tract tissues. High drug
concentrations of bronchial mucosa, alveolar macrophage, and epithelial lining fluid
indicate good penetration into the respiratory tract system.
The newer fluoroquinolones, including moxifloxacin, gatifloxacin, and gemifloxacin,

achieve bronchial mucosa and epithelial lining fluid concentrations in excess of the
minimum inhibitory concentration (MIC) values of most common respiratory tract
pathogens throughout substantial portions of their dosing intervals. Fluoroquinolones
have also shown impressive penetration into sputum and nasal tissue. Therefore, the good
penetration of fluoroquinolones into various respiratory tract tissues and fluid supports
one of several rationales for their utilization in respiratory tract infections.
11
Introduction
IV. Drug penetration into urinary tract system and reproductive system
Good tissue penetration of fluoroquinolones into kidney, prostate tissue, and gynecologic
tissue is well documented. This characteristic offers one justification for the use of
fluoroquinolones in the treatment of urinary tract or gynecologic infections.
V. Drug penetration into central nervous system
To date, no fluoroquinolones are FDA approved for the treatment of bacterial meningitis.
However, since fluoroquinolones achieve good penetration into cerebrospinal fluid
(CSF), there exists a potential for the development of fluoroquinolones for this indication.
Elimination
Most fluoroquinolones are eliminated primarily by hepatic elimination (metabolism or

biotransformation) and renal excretion. Trans intestinal elimination is an alternative mode
of elimination. Ciprofloxacin, levofloxacin, gatifloxacin, and gemifloxacin are highly
dependent on renal elimination.
Prolonged half-life is another significant characteristic of the newer-generation

fluoroquinolones. As listed in Table 1.1, the half-lives of levofloxacin, moxifloxacin and
gatifloxacin in healthy subjects are in the range of 8–16 hours. Because of their long half-
lives, these fluoroquinolones can be administered once daily. However, long half-life is
not always beneficial. It would be disadvantageous in patients who develop adverse
reactions to the drug.
Drug interactions
I. Drug–Food interaction
The oral absorption of some fluoroquinolones is delayed by the presence of food in the
gastrointestinal tract. Thus peak serum concentrations tend to be somewhat lower and the
12
Introduction
time to achieve peak concentrations is slightly delayed. This effect is clinically

insignificant. None of the fluoroquinolones are significantly affected by food. Since
absorption of all fluoroquinolones is inhibited by divalent cations, co-administration of
high-cation-content food products with fluoroquinolones should be avoided to promote
reliable administration.
II. Drug–Drug interactions
The absorption of all oral fluoroquinolones are affected by di- or trivalent-cation- (Mg 2+,
Al3+, or Ca2+) containing agents such as antacids, calcium, and iron supplements and
sucralfate. The mechanism of this interaction probably arises from the formation of
poorly absorbed chelated products consisting of fluoroquinolone molecules and metal
cations. In order to avoid the interaction between these cation-containing agents and
fluoroquinolones, the fluoroquinolones should be administered at least 4 hours before or
8 hours after cation-containing agents.
Therapeutic uses
The fluoroquinolones are effective in a number of bacterial infections and are prescribed
in the treatment of systemic and local diseases caused by a wide array of Gram-positive
and Gram-negative bacteria. Fluoroquinolones has shown a broad spectrum of
antibacterial activities, they have been advised in conditions such as prostatitis, deep-
seated infections, central nervous system infections, bone and joint infections, and
nosocomial infections resistant to other antibacterial drugs. Fluoroquinolone play an
active role in the treatment of a variety of severe infections that are either caused by
pathogens resistant to other antibiotics or infections that are located in tissues
inaccessible to antibiotics. Ciprofloxacin and norfloxacin have been extensively studied
and clinically evaluated. Norfloxacin has mostly been prescribed for the treatment of
urinary tract infections. In one study, it was observed that 408 out of 417 (98 %) gram-
negative isolates and 58 out of 62 (94 %) gram-positive isolates were vulnerable to
norfloxacin. Norfloxacin has great potency against pathogens that can be eliminated by
parenteral therapy only, and therefore, the whole spectrum of urinary tract pathogens can
13
Introduction
be dealt with a single oral dose of the drug. Therefore patients who once were required
for long-term hospitalization for parenteral therapy of complicated urinary tract infections
can now be discharged earlier and treated with the oral use of fluoroquinolones.
The usual dose of fluoroquinolones is continued for a maximum of 3 or 10 days.

Parenteral therapy should be given at changed sites when large volume and long dose of
drug is used. Injectable quinolones can cause indurations at the injection site.
Enrofloxacin has strong alkalinity and should be used with caution.
Side effects of fluoroquinolones [88-106]
It is generally more difficult to detect all the side effects of a drug than to prove its
effectiveness. Studies on the antimicrobial activity or clinical efficacy of an antibiotic are
comparatively easy because investigators know what they are looking for: inhibition of
microbes in vitro or clinical cure of an infectious disease. It is a planned search for an
expected result. However, searching for toxicity is open ended and must be performed
without definition of all possible endpoints.
Fluoroquinolones is a class of drugs which are relatively safe, well absorbed and well
tolerated. Compared to the curative effect and beneficial role of fluoroquinolones, their
side effects are not very severe with a few exceptions. Most of the adverse effects of
fluoroquinolones are self-controlled, mild in severity and rarely result in discontinuation
of the treatment. However, in a very few cases, serious adverse effects have been
observed. Some of the mild and common side effects include diarrhea, abdominal pain,
vomiting and nausea. Achilles tendonitis, phototoxicity (more common with
lomefloxacin and sparfloxacin) central nervous system effects (headache, confusion and
dizziness) and QT prolongation are some of the serious but less common adverse effects
of fluoroquinolones. Convulsion is very commonly associated with all drugs of this class.
The Food and Drug Administration has put this class of antibiotics in pregnancy risk
category C due to the possibilities to cause teratogenic or embryocidal effects.
Fluoroquinolones are prescribed for use only in adults (people older than 18).
Fluoroquinolones can retard the development of bones, teeth and cartilage in a child or
14
Introduction
fetus. These drugs are also passed over to breast milk and should not be advised to
mothers who feed their babies through breast.
Properties of drugs selected for this thesis [66, 67, 73, 78, 80, 105-128]
1.1 Ciprofloxacin
Ciprofloxacin is one of a new generation of fluorinated quinolones structurally related to

nalidixic acid. The primary mechanism of action of ciprofloxacin is inhibition of bacterial
DNA gyrase. It is a broad spectrum antibacterial drug to which most Gram-negative
bacteria are highly susceptible and many Gram-positive bacteria are susceptible or
moderately susceptible. Unlike most broad spectrum antibacterial drugs, ciprofloxacin is
effective after oral or intravenous administration. The results of clinical trials with orally
and intravenously administered ciprofloxacin have confirmed the potential for its use in a
wide range of infections, which was suggested by its in vitro antibacterial and
pharmacokinetic profiles. It has proven an effective treatment for many types of systemic
infections as well as for both acute and chronic infections of the urinary tract. The drug is
also well tolerated. Thus, as an orally active, broad spectrum and potent antibacterial
drug, ciprofloxacin offers a valuable alternative to broad spectrum parenterally
administered antibacterial drugs for use in a wide range of clinical infections, including
difficult infections due to multiresistant pathogens.
1.1.1 Physical characteristics
Molecular formula: C17H18FN3O3
Molecular mass: 331.346 g/mol
Structural formula: The structure formula of ciprofloxacin is given in figure 1.1
15
Introduction
Figure 1.1 Structural formula of ciprofloxacin
Systematic (IUPAC) name: 1-cyclopropyl-6-fluoro-4-oxo-7-piperazine-1-yl-quinoline-

3-carboxilic acid.
Appearance: Ciprofloxacin is white or slightly yellow crystalline powder.
Melting point: 257 oC
Solubility: Slightly soluble in water (1.1 mg/L) and ethanol and highly soluble in
0.1N hydrochloric acid.
1.1.2 Dosage and mode of administration
Normally, ciprofloxacin is taken at a dose of 250 – 500 mg two times a day, after every
12 hours. The therapeutic doses of ciprofloxacin range from 250 to 750 mg, after every
12 hours. The duration of the therapy depends on the severity of the infections, clinical
progress and microbiological response. Normally the medication is continued for 2 – 3
days after normalization of temperature and fading of indications. The span of medication
is normally 7 – 14 days, however it can be extended to 4 – 6 weeks in case of infection in
joints and bones. Reduced dose is administered in individuals with weakened renal
function. Ciprofloxacin in tablet dosage form is swallowed unchewed with sufficient
amount of liquid and can be taken either before or after meals. The course must be
completed and should not be discontinued after normalization of temperature or fading of
16
Introduction
symptoms. The therapy can be stopped only noticing the adverse effects or lack of
improvement. If the dose is missed, it should be taken at the earliest convenient and
administering double dose should always be avoided.
Ciprofloxacin is available in tablets, solution, suspension and ointment formulations. It is

administered orally and intravenously. In the form of ointment, it is also applied
topically.
1.1.3 Over dosage
In case of intoxication with ciprofloxacin, no specific antidote is available. The

immediate procedure is to make the stomach empty. It can be done by vomiting or gastric
lavage. It will help in reduction of further absorption of the drug. It is also essential to
provide the patient with adequate hydration and symptomatic treatment.
1.2 Sparfloxacin
Sparfloxacin is a recently developed fluoroquinolone. It is a broad spectrum antibacterial

fluoroquinolone active against some microorganisms including Gram-positive and Gram-
negative bacteria and demonstrates moderate activity against anaerobes and
Mycobacteria, for which the quinolones in general have low activity. It is used in the
treatment of bacterial infections. Sparfloxacin exerts its antibacterial activity by
inhibiting DNA gyrase, a bacterial topoisomerase. DNA gyrase is an essential enzyme
which controls DNA topology and assists in DNA replication, repair, deactivation, and
transcription.
Molecular formula: C19H22F2N4O3
17
Introduction
Figure 1.2 Structural formula of sparfloxacin
Systematic (IUPAC) name: 5-amino-1-cyclopropyl-7-[(3R,5S)3,5-dimethypiperazine-1-

yl]6,8-difluoro-4-oxo quinoline-3-carboxilic acid.
Appearance: Sparfloxacin is yellow crystalline solid.
Melting point: 267 oC
Density: 1.436 g/cm3
Solubility: Sparfloxacin is insoluble in water while soluble in almost all organic

solvents, such as methanol, ethanol, acetone, cyanomethane etc.
Dosage form: It is available in film coated tablets form containing 100 mg and 200 mg
of sparfloxacin per tablet. Its route of administration is oral.
1.3 Levofloxacin
Levofloxacin is a second generation fluoroquinolone and is a levorotatory isomer of

Ofloxacin. It has shown excellent activity against a variety of Gram-positive and Gram-
negative organism which are resistant to the established agents. Its DNA gyrase activity
is similar to that of ciprofloxacin but concentrations of levofloxacin in serum and tissues
18
Introduction
exceed those of ciprofloxacin, and the half-life of levofloxacin is longer, allowing daily
single-dose administration in many cases.
Figure 1.3 Structural formula of Levofloxacin
Systematic (IUPAC) name: (S)-7-fluoro-2-methyl-6-(4-methylpiperazin-1-yl)-10-oxo-

4-oxa-1-azatricyclo[7.3.1.0{5,13}]trideca-5,7,9(13),11-tetraene-11-carboxylic acid
Appearance: Levofloxacin is off white or slightly yellow crystalline powder.
Melting point: 225 – 227 oC
Solubility: Levofloxacin is insoluble in water and is soluble to free soluble at acidic

pH while solubility decrease above pH 6.7.
19
Introduction
The usual dose of levofloxacin tablets or oral solution is 250 mg, 500 mg, or 750 mg
administered orally every 24 hours, as indicated by infection. The usual dose of
levofloxacin injection is 250 mg or 500 mg administered by slow infusion over 60
minutes every 24 hours or 750 mg administered by slow infusion over 90 minutes every
24 hours, as indicated by infection.
These recommendations apply to patients with creatinine clearance ≥ 50 mL/min. For

patients with impaired renal function and with creatinine clearance < 50 mL/min, the
dosing regimen is reduced and adjusted. Severity of infections, microbial response and
clinical success determine the duration of therapy. Normally, the treatment with
levofloxacin continues 2 – 3 days after normalization of temperature and disappearance
of symptoms. Levofloxacin is advised normally for 7 – 14 days, however, the therapy can
be continued for 4 – 6 weeks in case of severe infections in bones and joints.
Levofloxacin in the form of tablets dosage form is swallowed unchewed with sufficient
amount of liquid and can be taken either before or after meals. The course must be
completed and should not be discontinued after normalization of temperature or fading of
symptoms. The therapy can be stopped only observing the adverse effects or lack of
improvement. If the dose is missed, it should be taken at the earliest convenience and
administering double dose must always be avoided.
Levofloxacin is available in tablets, solution, and suspension and eye drops formulations.
It is administered orally and intravenously.
1.3.3 Over dosage
In case of overdosing, no particular antidote is available for levofloxacin. In the event of

an acute over dosage, the stomach should be emptied. The patient should be observed and
appropriate hydration maintained. Levofloxacin is not efficiently removed by
hemodialysis or peritoneal dialysis.
20
Introduction
1.4 Gatifloxacin
Gatifloxacin is a manmade antibiotic and member of fluoroquinolone family. It is the

fourth-generation antibiotic of the family. It is bactericidal and inhibits activity of
bacterial enzymes DNA gyrase and topoisomerase IV and thus stops replications of
bacteria. Gatifloxacin is the research product of Bristol-Myers Squibb and was introduced
in 1999 under the proprietary name Tequin® for the treatment of respiratory tract
infections, having licensed the medication from Kyorin Pharmaceutical Company of
Japan. Allergan produces an eye-drop formulation called Zymar®. Gatifloxacin was
available in tablets form and in various aqueous solutions for intravenous therapy.
Figure 1.4 Structural formula of Gatifloxacin
21
Introduction
Systematic (IUPAC) name: 1-cyclopropyl-6-fluoro- 8-methoxy-7-(3-methylpiperazin-

1-yl)- 4-oxo-quinoline-3-carboxylic acid
Appearance: Gatifloxacin is pale yellow to crystalline powder.
Solubility: Solubility of gatifloxacin is pH dependent, with maximum aqueous

solubility (40 – 60 mg/mL) occurring in a pH range of 2 – 5.
Gatifloxacin was approved for the treatment of acute sinusitis, community-acquired

pneumonia, complicated and uncomplicated urinary tract infections, pyelonephritis,
uncomplicated urethral and cervical gonococcal infections, and acute uncomplicated
rectal gonococcal infections in women caused by susceptible pathogens. The most
commonly recommended dosage was 400 mg once/day orally or intravenously by slow
infusion over 60 minutes for 7-10 days. Uncomplicated urinary tract infections were
treated with a single oral dose of 400 mg or a 3-day regimen of 200 mg/day. A dosage
reduction was recommended in patients with creatinine clearances below 40 mL/minute,
including those receiving hemo-dialysis.
The drug was available as 200 and 400 mg oral tablets. The injectable form was available
as 200 mg (10 mg/mL) and 400 mg (10 mg/mL) single-use vials or premixed bags. In
most part of the world, manufacturing, distribution and sale of gatifloxacin (oral and
injectable) has been banned due to its certain life threating side effects. This medicine is
currently available only as an eye solution.
1.4.3 Over dosage
If overdose is suspected, local poison control center or emergency room should be

contacted immediately. In case of over dosage in ophthalmic solution, eyes may be rinsed
with warm water. An overdose of gatifloxacin ophthalmic is not likely to cause life-
threatening symptoms.
22
Introduction
1.5 Moxifloxacin
Moxifloxacin is a fourth-generation synthetic fluoroquinolone antibacterial agent

developed by Bayer AG (initially called BAY 12-8039). It is marketed worldwide (in the
hydrochloride form) under different brand names for oral treatment. In most countries,
the drug is also available in parenteral form for intravenous infusion. Moxifloxacin is also
sold in an ophthalmic solution (eye drops) for the treatment of conjunctivitis (pink eye).
Moxifloxacin is a broad spectrum, 8-methoxyquinolone antimicrobial approved by the
Food and Drug Administration in December 1999 for use in the treatment of acute
bacterial sinusitis, acute bacterial exacerbations of chronic bronchitis, and community-
acquired pneumonia caused by susceptible microorganisms. This antimicrobial agent
displays excellent activity against common community-acquired respiratory pathogens,
such as Streptococcus pneumoniae, Haemophilus influenzae, Moraxella catarrhalis,
Mycoplasma pneumoniae, and Chlamydia pneumoniae. Moxifloxacin is also highly
active against other pathogens, such as methicillin-susceptible Staphylococcus aureus
(MSSA) and Klebsiella pneumoniae. It is also used to treat bacterial infection in the eyes.
23
Introduction
Figure 1.5 Structural formula of Moxifloxacin
Systematic (IUPAC) name: 1-cyclopropyl-7-[(1S,6S)-2,8-diazabicyclo [4.3.0]non-8-

yl]-6 fluoro-8-methoxy-4-oxo- quinoline-3-carboxylic acid
Appearance: Moxifloxacin is off white to yellow powder.
Solubility: Moxifloxacin is soluble in 0.1 mol/L sodium hydroxide. It is sparingly
soluble in water and methanol, and slightly soluble in 0.1 mol/L hydrochloric acid and
ethanol.
1.5.2 Moxifloxacin dosing guidelines
Commercially, it is available as ophthalmic solutions, oral tablets and intravenous

infusions. Normally single dose of 400 mg of moxifloxacin once a day is prescribed. The
medication should not be interrupted even if one starts getting better and should be
continued for 2 – 3 more days. Possible bacterial resistance to the antibiotic can be
developed in case of stopping the medication earlier. The length of time of the therapy
will depend on the kind and severity of infections. In case of acute infections, the
medication is prescribed for as few as five days while chronic and severe infections
require much longer use of the drug.
24
Introduction
1.5.3 Over dosage
In case of acute overdose of moxifloxacin, the stomach should be emptied through

vomiting or gastric lavage and adequate hydration of the patient should be maintained.
For possible prolongation of QT interval, ECG monitoring is recommended. The patient
should be put on careful observation and symptomatic and supportive treatment can be
given. The administration of activated charcoal immediately after oral overdose may
block excessive increase of systemic moxifloxacin exposure. About 3 % and 9 % of the
dose of moxifloxacin, as well as about 2 % and 4.5 % of its glucuronide metabolite can
be removed by continuous ambulatory peritoneal dialysis and hemodialysis, respectively.
25
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References
39
LITERATURE REVIEW
Literature Review
Vandana and Chaudhary (1) reported a simple high pressure thin layer chromatographic
method with good accuracy, precision and reproducible results for the regular estimation
of moxifloxacin dosage formulations. The method was evaluated according to directives
of International Conference on Harmonization. Making the use of silica gel 60 F 254 as
solid phase and a mixture of dichloromethane, methyl alcohol, strong aqueous ammonia
and cyanomethane (10:10:5:10) as mobile phase, moxifloxacin was determined at 292 nm
using densitometry. The signal versus concentration response was linear from 9-54
nanograms. Correlation coefficient of the proposed method was found to be 0.99.
Vandana and Chaudhary (2) reported a new, simple and economical spectrophotometric
method for the quantification of moxifloxacin hydrochloride in the marketed
formulations. The method was found accurate, precise and sensitive having good linear
correlation between drug concentration and absorbance. International Conference on
Harmonization and United State Pharmacopeia guidelines were used for the confirmation
of various parameters of the method. Deionized water was used during the assay and
absorption of the drug was measured at 294 nm. Molar absorptivity of the drug was found
4.3166 x 104 L/mol/cm and regression equation was established as, absorbance = 0.0933
x concentration in µg/mL + 0.0012 (R2 = 0.9998).
Sultana et al (3) reported an simple reversed phase HPLC method for the quantification
of moxifloxacin in raw material, marketed products and serum. Two C 18 columns
(Purospher STAR C18 and Discovery C18) were tested having the same dimensions (25
cm x 4.6 mm x 5 µm) with mixture of methyl alcohol, cyanomethane and water (85:5:10)
as a mobile phase. Phosphoric acid was used for pH adjustment (2.75) of the medium.
The mobile phase was run at the rate of 1 mL/min, while UV detector at 290 nm. The
method was reported to be precise (intraday difference 0.04-0.86 % and interday
difference 0.12-0.94 %), accurate (99.39-102.67 %) and specific. Linear calibration curve
was plotted in the range of 0.39-25 µg/mL with 0.999 as correlation coefficient. Limit of
detection and quantitation was 0.002-0.51 µg/mL and 0.01-0.555 µg/mL respectively.
The results were verified with the help of student’s t-test and analysis of variance. It was
suggested that the proposed method could be employed for the routine quantification of
moxifloxacin in marketed products and blood samples of human beings.
38
Literature Review
Radi et al. (4) described an electroanalytical technique for quantitation of moxifloxacin,

sparfloxacin and gatifloxacin. Glassy carbon electrode with a wide variety of buffers of
different pH was tried. Potential range of 0.65 – 1.1 V vs Ag-AgCl-KCl was used for the
electrochemical oxidation of the drugs. The drugs were oxidized irreversibly and showed
adsorption-controlled process behavior at all pH and buffers under consideration.
Adsorption behavior of the drugs on glassy carbon electrode made the basis of this
electroanalytical method. Relationship between peak current and concentration of the
drugs was linear. The three fluoroquinolones were successfully determined in bulk and
pills using differential pulse voltammetry. Results of the method were statistically
compared with spectrophotometric methods for the drugs.
Sultan (5) presented a kinetic spectrophotometric method for determination of

moxifloxacin in raw material and marketed formulations. A coloured product was
obtained by treating moxifloxacin with chloranil in the presence of ethanal. The coloured
product was monitored at 652 nm. Optimum conditions for the reaction were
investigated. Reaction mechanism was proposed and stoichiometric ratio among the
reactants was determined. Kinetic study of the reaction was conducted and activation
energy was found to be 6.65 KJ/mol. Calibration curve was drawn under optimized
conditions by two ways. One way was initial rate and second was flat time of five
minutes. The calibration curves were linear in the range of 4 – 100 µg/mL and 15 -150
µg/mL for the methods respectively. Detection limit for initial rate method was found to
be 2.0 µg/mL while it was 5.0 µg/mL for flat time method. Validation of the method with
respect of analytical performance was carried out and was found acceptable. The method
was found free of interferences of the common excipients added in marketed
formulations. The method was applied for determination of moxifloxacin in the marketed
dosage forms and percent recovery with the label value was 101.25 ± 0.73 for initial rate
method and 100.92 ± 0.65 for flat time method. Results were statistically analyzed with
standard spectrophotometric methods and a good similarity in accuracy and precision of
the two methods was observed. The methods were found to be of great importance and
applicable for the analysis of moxifloxacin in QC labs.
39
Literature Review
Djurdjvic et al. (6) proposed a method with the help of DryLab ® software and
chemometric (response surface) approach for the separation and quantification of
impurities of moxifloxacin in dosage forms and for monitoring of degradation products.
Four synthesis related impurities were separated with the help of Waters C 18 XTerra
column using mixture of 2 % aqueous tri ethyl amine and cyanomethane (90:10) as
mobile phase. Phosphoric acid was used for pH (6.0) adjustment. Mobile phase was run
at the speed of 1.5 mL/min and the analyte was detected at 290 nm, keeping column
temperature at 45 oC. Average resolution between the two closed peaks for impurities was
greater than 1.5. Calibration curve was linear in the range of 0.2 - 2.0 µg/mL with
detection limit of 0.05 µg/mL and quantification limit of 0.20 µg/mL. The proposed
method is suitable for finding impurities in marketed formulation and the study of
degradation products in stability study of the drug. The total impurities in the marketed
dosage forms was found to be 0.1 % of the total drug and two degradation products were
found in hydrolytic condition. The method is fit for accurate and fast determination of
moxifloxacin hydrochloride in its tablets stability study.
Kumar and Ramachandran (7) reported a HPLC method for the determination of
moxifloxacin in human plasma. Deproteination of the sample was done with perchloric
acid. The supernatant was analyzed using RP C 18 column with fluorimetric detection at
290 nm as excitation and 460 nm as emission wavelength. The method gives a linear
response from 0.125-10.0 µg/mL and the method was found specific for moxifloxacin.
Less than 10 % coefficient of variation for intra and inter day analysis was reported. The
average recovery of moxifloxacin from the samples was 101 %. The method was dimple
and could be employed for pharmacokinetics study of moxifloxacin.
De Smet et al. (8) described a high performance liquid chromatographic method for
determination of Ofloxacin, ciprofloxacin and moxifloxacin in blood samples of human
being in a single run. Sarafloxacin was used as internal standard in the method. Waters
XBridgeTM C18 column with cyanomethane, methanol, 0.025M tetra butyl ammonium
chloride and tetra fluro acetic acid in the ratio of 75:25:899:1 as mobile phase A and in
the proportion of 150:50:799:1 as mobile phase B was used. Excitation wavelength and
emission wavelength for ciprofloxacin were 279 nm and 442 nm respectively and 290 nm
40
Literature Review
and 500 nm for the rest of analytes. Plasma samples were deproteinated by treating with
cyanomethane and the liquid layer was evaporated and the samples free from protein
were reconstituted in mixture A. The method response was linear for the three drugs over
the clinically matched concentration range of 0.02-7.50 µg/mL, with good correlation
coefficient (greater than 0.995), high precision (coefficient of variation ≤ 7 %), and 90.4 -
105.4 % accuracy. Limit of quantification of the drugs was found to be 0.02 µg/mL. For
Ofloxacin recovery from plasma was 95 % while mean recovery of ciprofloxacin and
moxifloxacin was 86.4 % and 94.2 % respectively. Signal of the method was stable after
three freeze-thaw cycles and for minimum fifteen hours. The method was applied for the
analysis of the samples obtained from patients participating in pharmacokinetics study of
moxifloxacin.
Ulu (9) reported a simple, fast and highly sensitive spectrofluorimetric method for the
quantification of moxifloxacin, norfloxacin, ciprofloxacin and enoxacin in marketed
dosage forms. The indirect determination of the four quinolones was based on reaction of
the drugs with 4-chloro-7-nitrobenzofurazan in borate buffer of pH 9.0, leading to the
formation of yellow coloured product. Experimental parameters were thoroughly studied
and optimized. Signal versus concentration was linear in the range of 33.5 – 1000 ng/mL
for moxifloxacin, 29.5 – 800 ng/mL for norfloxacin, 28.5 – 700 ng/mL for enoxacin and
23.5 – 500 ng/mL for ciprofloxacin. As low as 9.98 ng/mL, 9.2 ng/mL, 8.5 ng/mL and
7.0 ng/m of moxifloxacin, norfloxacin, enoxacin and ciprofloxacin respectively can be
determined by the proposed method. Data for inter and intraday analysis was recorded.
High precision of the proposed method was inferred from low coefficient of variation and
good percent recovery values proved accuracy of the method. The method was specific
and highly sensitive. The results obtained were in agreement with that of the reference
and official methods. The proposed spectrofluorimetric method is applicable for the
quantification of the four fluoroquinolones in pharmaceutical dosage forms and the
method is free from the interferences of common excipients used in the formulations.
Faria et al. (10) proposed a capillary zone electrophoresis technique for the quantification
of four fluoroquinolones. A plain aqueous electrolyte system of 25 mmol/L of tris
(hydroxymethyl) aminomethane/HCl and 15 mmol/L of sodium tetra borate buffer of pH
41
Literature Review
8.87 with UV detection at 282 nm was used in the technique. Correlation coefficient of
the technique was almost one, which showed linearity of the method. Slope of the
calibration curve for external standard and slope of the calibration curve for standard
addition were comparable which showed selectivity of the method. Percent coefficient of
variation was less than 3.94, 3.87, 1.30 and 1.88 for ciprofloxacin, gatifloxacin,
moxifloxacin and ofloxacin respectively which showed reproducibility of the method.
The method was accurate and the mean percent recovery was 101.2 for ciprofloxacin,
101.0 for gatifloxacin, 101.3 for moxifloxacin and 99.9 for ofloxacin. Detecting limit
was 2.72 µg/mL for ciprofloxacin, 1.92 µg/mL for gatifloxacin, 0.795 µg/mL for
moxifloxacin and 1.05 µg/mL for ofloxacin. Limit of quantification was 9.06 µg/mL for
ciprofloxacin, 6.40 µg/mL for gatifloxacin, 2.65 µg/mL for moxifloxacin and 3.50 µg/mL
for ofloxacin. The method was checked for its robustness. Due to the ease of use,
selectivity, accuracy, precision and speed, it is fit for analysis in quality control labs in
pharmaceutical industries.
Nemutlu et al. (11) reported RP-HPLC for simultaneous separation and quantification of
seven fluoroquinolones in amniotic fluids and blood plasma. Optimum experimental
conditions for maximum resolution and minimum retention time were evaluated by
applying experimental design to the method. Two different variables were optimized
together with the help of total desirability function D. The experimental parameters were
put in the second order polynomial. Mobile phase consisting of 15 mM citrate buffer, 9 %
cyanomethane, 5 % methanol and 5 mM tetra methyl ammonium bromide maintained at
pH 3.2 was run using flow rate 1.5 mL/min, with column temperature maintained at
40oC. Samples were prepared by an easy and effective solid phase extraction. More than
95 % of the drugs were recovered. Specificity accuracy, precision, linearity and
robustness of the method were checked and the optimized experimental parameters were
confirmed according to FDA plan. The method was applied for determination of
moxifloxacin and levofloxacin in plasma and amniotic fluids.
Karim et al. (12) reported a batch chemiluminescence determination method for

moxifloxacin. The method is based on the enhancement of chemiluminescence emission
of Ru (bpy)32+ - Ce (IV) system in the presence of moxifloxacin. Enhancement effect was
42
Literature Review
proportionate to the concentration of moxifloxacin and is linear in the range of 1.0 x 10 -6

M to 1.0 x 10-4 M under optimized working conditions. As low as 3.0 x 10 -7 M of the
drug could be detected by the method. Coefficient of variation of the method was as low
as 1.75 % for ten replicates.
İnam et al. (13) reported a sensitive, fast and simple differential pulse polarographic
method for the assay of moxifloxacin in dosage forms and biological samples.
Irreversible cathodic peak of moxifloxacin was detected in Britton Robinson buffer
system of pH range 5 – 11. At optimum pH 10, using saturated Calomel electrode, a well-
defined peak was achieved. Calibration curve was linear in the concentration range of 5 x
10-7 – 1 x 10-4 M with 0.995 as correlation coefficient for ten readings. The diffusion
current constant was 1.48 ± 0.12. The method was applied for analysis of pharmaceutical
formulations and the results were almost same as that of the spectrophotometric method.
The method was useful for determination of moxifloxacin in spiked samples of biological
nature such as human serum and urine.
Ciric et al. (14) reported a simple, fast and reliable spectrophotometric method for
determination of moxifloxacin in human plasma samples. Citrate-phosphate buffer of pH
7.2 in the presence of sodium dodecylsulfate (12 mmol/L) was used as medium of the
analysis. Moxifloxacin was determined by measuring peak to peak height in the
wavelength range of 335 – 345 nm using second order derivative spectra. Calibration
curve was linear in the concentration range of 0.25 – 10 µg/mL with 0.03 µg/mL as
detection limit of the method. Percent recovery of the method was 95 - 102. Sensitivity of
the method was improved by adding surfactant. Analysis of plasma of healthy volunteers
was carried out successfully. Validation and confirmation of the results of the method
was done with newly developed HPLC method.
Trindade et al. (15) reported an electroanalytical technique for determination of

moxifloxacin in dosage forms. The technique is based on complexation of the drug with
cuprous ion followed by electrochemical reduction at hanging mercury drop electrode.
Using square wave voltammetry, a very clear reduction peak of the complex at – 0.21 V
using Silver/Silver chloride as reference electrod in phosphate buffer of pH 8.0 was
recorded. Accumulation time of ten seconds at – 0.4 V was used in the analysis and
43
Literature Review
detection limit was found to be 3.6 x 10-8 mol/L. A spectrophotometric method was used
as comparison method and the results were in agreement with each other.
Motwani et al. (16) reported new, precise and reliable methods for determination of
moxifloxacin in mass and dosage forms. Absorption was monitored at 296 nm and 289
nm in 0.1 N HCl medium (pH 1.2) and phosphate buffer medium (pH 7.4) respectively.
Absorption as a function of concentration was proportional in concentration range of 1 –
12 µg/mL in acidic medium and 1 – 14 µg/mL in basic medium with correlation
coefficient of 0.9999 and 0.9998 respectively. The method is sensitive having molar
absorptivity and Sandell’s sensitivity coefficient as 4.63 x 10 4 L/mol/cm and 0.5
ng/cm2/0.001A in acidic medium and 4.08 x 10 4 L/mol/cm and 10.8 ng/cm2/0.001A in
basic medium respectively. ICH instructions were used for checking and confirmation of
various parameters of the methods. Limit of detection and quantification of the methods
in acidic and basic medium were found to be 0.0402 µg/mL and 0.1217 µg/mL and
0.0384 µg/mL and 0.1163 µg/mL respectively. Moxifloxacin was successfully
determined in pharmaceutical products by the proposed methods. Coefficient of variation
of the methods was less than two percent which showed reproducibility of the methods.
The methods were found simple, inexpensive and less time consuming and could best be
applied for quantification of moxifloxacin in marketed formulations and other samples.
Laban-Djurdjvic et al. (17) reported a simple and fast reversed phase high performance
liquid chromatographic method for the direct quantification of moxifloxacin in blood
samples of human beings. Supelco LC-Hisep shielded hydrophobic phase column was
used for the separation of moxifloxacin from plasma components. A mixture of
cyanomethane and 0.25 M sodium phosphate buffer of pH 3 in the proportion of 5:95
with a flow rate of 1 mL/min was used as mobile phase. Excitation wavelength 290 nm
and emission wavelength 500 nm was used for fluorometric detection. Samples were
prepared simply by adding sodium dodecysulfate, ofloxacin and dilution with water. The
best possible experimental conditions were set up by the response surface method in two
factor space (dependence on retention time on percent volume of cyanomethane and on
pH of mobile phase). The response was linear in the range of 3-1300 µg/L with good
correlation coefficient (0.99986). Average recovery of the method was found to be
44
Literature Review
92.5 % while detection and quantification limits were 1 and 3 µg/L respectively. The
method was applied for the quantification of moxifloxacin in human plasma after its oral
administration in the form of single or repeated doses of Avelox ® tablets. Due to its speed
and accuracy, pharmacokinetics studies and routine practices in clinics can be carried out
with the proposed method.
Ulu (18) presented a high performance liquid chromatography method for quantification
of moxifloxacin in blood samples of human beings using fluorescence detection with
norfloxacin as internal standard. Pre-column derivatization of moxifloxacin and internal
standard were prepared with 4-chloro-7-nitobenzodioxazole. C 18 column and mixture of
cyanomethane and 10 mM orthophosphoric acid (pH 2.5) in the ratio of 80 to 20 was
used as mobile phase. The reaction product is fluorescent active having 464 nm excitation
wavelength and 537 nm emission wavelength. Linearity of the method in human blood
samples was in the range of 15-2700 ng/mL with good reproducibility. The detection and
quantification limits of the method were 6 ng/mL and 15 ng/mL respectively and the
method was found sensitive. The proposed method was used for the pharmacokinetics
study of moxifloxacin in healthy volunteers.
Schulte et al. (19) reported a sensitive, precise, accurate and simple reversed phase high
performance liquid chromatographic technique with fluorimetric detection for
determination of moxifloxacin and levofloxacin. Levofloxacin was used as internal
standard for moxifloxacin while moxifloxacin was used as internal standard for
levofloxacin. The drugs were extracted from human plasma by a single step liquid-liquid
extraction. Calibration curve was linear in the range of 0.1-15 µg/mL for levofloxacin
and 0.2-7 µg/mL for moxifloxacin. Levofloxacin and moxifloxacin can be determined at
concentration level of 0.05 µg/mL and 0.2 µg/mL respectively by the proposed method.
Volunteers taking moxifloxacin or levofloxacin as single dose at two different times were
subjected for studying of plasma drug concentration through the proposed technique.
Trindade et al. (20) reported a simple, fast, reliable and sensitive square wave
voltammetric method for quantification of moxifloxacin in marketed formulations and
spiked samples of human urine. The method is based on the electrochemical reduction of
the analyte on hanging mercury drop electrode using phosphate buffer (0.04 mol/L) of pH
45
Literature Review
8.0 as support electrolyte. At optimized experimental conditions; reduction peak of the

analyte was obtained at – 1.38 V versus Silver/Silver chloride. Limit of detection for pure
and dosage forms was 0.44 and 3.20 ng/mL respectively. Similarly limit of quantification
was 1.46 and 10.60 ng/mL respectively. The method was applied to marketed
formulations and spiked urine samples. The method was found more sensitive than
spectrophotometric and spectrofluorimetric methods having same accuracy and precision.
Ocana-Gonzalez et al. (21) investigated a liquid chromatographic method for

simultaneous determination of quinolones and cephalosporin in human urine. C 18 column,
mobile phase containing cyanomethane, n-octyl amine in phosphoric acid (0.1M) and
sodium hydroxide buffer system (pH 3) with UV diode array detector were used.
Injection volume for the sample was 20 µL and flow rate was maintained at 1 mL/min.
Detection wavelengths were 294 nm, 292 nm, 282 nm and 256 nm for moxifloxacin,
levofloxacin, garenoxacin and cefepime respectively. Retention time and detection limits
for garenoxacin, moxifloxacin, levofloxacin and cefepime were 10.7, 8.9, 7.5 and 4.9 min
and 1.8, 1.9, 2.2, and 2.7 µg/mL respectively. The proposed method was applicable for
the separation and quantification of the four antibiotics in spiked urine samples.
Guerra et al (22) reported a microbiological assay and liquid chromatographic methods

for determination of moxifloxacin in solid dosage forms. The bioassay method involved a
cylinder-plate agar diffusion using Micrococcus luteus ATCC 9341 as the test
microorganism while as a diluent 0.1M phosphate buffer solution of pH 8.0 was used.
Linear curves both for standard and test solution were obtained with 0.9479 as correlation
coefficient. In the tested level of drug concentration 4.0, 8.0 and 16.0 µg/mL, no
parallelism deviation in the curves was observed. Percent recovery was in the range
96.25-100.5 and 2.73 % was interday precision. LC analyses were carried out using
Shim-Pack CLC-ODS column having 250 x 4.6 mm x 5µm dimensions and mixture of
0.17 % phosphoric acid with 0.05M tetramethylammonium hydroxide and cyanomethane
(95:5) as mobile phase A and mixture of 0.17 % phosphoric acid with 0.05 M
tetramethylammonium hydroxide and methanol (55:45) as mobile phase B. pH of the
medium was set as 3.0. Flow rate of the mobile phase was maintained at 1 mL/min. The
analyte was monitored at 294 nm. Linear response was in the range 12-42 µg/mL with r
46
Literature Review
equal to 0.9999 and interday precision was found to be 1.93 %. Percent recovery was in
the range of 101.9-103.81. Active ingredients of moxifloxacin in tablets exposed to UV
radiation were estimated by both the methods and matching results were obtained.
Ferdig et al. (23) described high performance liquid chromatographic method for
determination of nine fluoroquinolones in environmental samples. C 18 bespoke silica was
used as solid phase and mixture of 50 mM formic acid and methanol as mobile phase
which was found fully compatible with MS coupling. Fluorimetric detector or MS using
the modes of SIM and selected reaction monitoring (SRM) were used in the proposed
method. Fluorimetric quantification limit was in the range of 10 – 60 µg/L while
detection with MS gave better limit of quantification. SPE (preconcentration factor of
1000) was employed for cleanup and preconcentration of the sample which took the limit
of quantification to ng/L range.
Nguyen et al (24) reported liquid chromatographic method with column switching facility
for the direct determination of moxifloxacin, gatifloxacin and levofloxacin in human
blood samples in a single run. Serum samples were loaded on pre column LiChroCart ® 4-
4 filled with a LiChrospher ® 100 RP-18, 5 µm where purification and preconcentration of
the drugs took place. The fluoroquinolones were moved on to analytical column
Supelcosil ABZ Plus (150 x4.6 mm i.d) for separation using mixture of phosphate buffer
10 mM (pH 2.5), cyanomethane (88:12) and tetra butyl ammonium bromide as mobile
phase. For maximum separation of the drugs, experimental parameters such as buffer
type, pH and concentration of cyanomethane in the mobile phase, ion pair reagents were
thoroughly investigated. With 5 µL samples injection volume and fluorimetric detection,
125 ng/mL was found limit of quantification for moxifloxacin and levofloxacin and 162.5
ng/mL for gatifloxacin. Time demanding processes are not required in the on line
technique and has short run time. The method is accurate, precise, linear, selective and
has good percent recovery and is suitable for routine determination and pharmacokinetics
studies of the investigated drugs.
Erk (25) presented an electroanalytical assay for the quantitation of moxifloxacin.

Different oxidation modes of moxifloxacin at glassy carbon electrode were studied. A
number of buffer systems were tried and variety of electroanalytical techniques such as
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cyclic voltammetry, differential pulse polarography and Osteryoung square wave

voltammetry were employed. Britton Robinson buffer of pH 6.0 was selected for the
analysis. Moxifloxacin could be oxidized irreversibly over the pH range of 2 – 10 and the
process was found diffusion controlled. The method was linear in the concentration range
of 4.47 x 10-7 to 1.0 x 10-5 mol/L and was successfully applied for the determination of
moxifloxacin in pills and human plasma. In addition a high performance liquid
chromatographic method was also proposed. In this method diode array detection system
was employed. Calibration curve was linear in the concentration range of 4.0 x 10 -6 to 5.0
x 10-5 mol/L moxifloxacin. Application of the methods for determination moxifloxacin in
human plasma and pharmaceutical formulations was accurate and precise. The results
were statistically treated and found satisfactory.
Vishwanathan et al. (26) reported a selective, sensitive and fast method for determination
of moxifloxacin in blood plasma. The method is based on liquid chromatography electro
spray ionization coupled with mass spectrometry. Lomefloxacin was used as internal
standard in the method. The analyte and the internal standard were extracted from blood
plasma using Oasis® HLB column. Triple quadrupole mass spectrometer with positive
electro spray ionization in the multiple reaction monitoring modes was used to monitor
the precursor and product ions. Mechanisms were proposed for the formation of expected
collision induced dissociation products of moxifloxacin. Calibration curve was linear in
the range of 1-1000 ng/mL with correlation coefficient greater than 0.999. Percent
coefficient of variation of intraday and interday results was less than 11.3 while percent
error of the method was also less than 10. Limit of detection of the method was found to
be 50 pg/mL.
Liang et al. (27) reported an accurate, sensitive and selective liquid chromatographic
method coupled with fluorescence and ultraviolet detectors for determination of
fluoroquinolones in human blood samples. Six quinolones, moxifloxacin, levofloxacin,
gatifloxacin, ciprofloxacin, trovafloxacin and cinoxacin were separated by the method
using C18 column. Experimental parameter such as concentration of cyanomethane,
buffers, pH, ion-pair and competing base reagents and composition of mobile phase were
optimized. Samples were prepared by adding internal standard and displacing reagent and
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processed by ultra-filtration technique. Spiked samples of human plasma were used for
evaluating accuracy, precision, linearity, recovery and selectivity of the proposed method.
Pharmacokinetics of levofloxacin was successfully studied by application of the method
to real samples.
Kudo et al. (28) reported a high performance liquid chromatographic technique for
determination of six new quinolones in blood plasma. After removing high molecular
weight proteins from the plasma samples using Molcut II membrane filter, they were
directly loaded to a HPLC column. Separation of the new quinolones present in the
filtrate was carried out on pre column using octadecylsilane solid phase and was then
loaded onto analytical column containing same solid phase material by column switching.
The studied drugs were detected by monitoring absorbance in ultraviolet region in the
range 269-300 nm. Calibration curve was linear in the range of 50 – 4000 ng/mL and 20
ng/mL was found to be detection limit of the technique. Quinolones added to the plasma
were recovered in the percent range 96.1 - 101.4 with coefficient of variation less than
5 %. Drugs level in plasma of patients can be determined and concentration of the drugs
in healthy volunteers for pharmacokinetics studies can be monitored by the proposed
method. Interferences study of the metallic ions such as Mg 2+, Al3+ and Fe3+ was also
carried out. Concentration of ofloxacin in skin tissue in the patients being treated with the
drug was also determined by the method. A good correlation coefficient (0.84) was
obtained in serum level and skin tissue level of ofloxacin in the study of thirty patients
being treated with ofloxacin.
Ocana et al. (29) reported a spectrofluorimetric method for determination of moxifloxacin

in marketed dosage forms, human blood and urine samples. Phosphoric acid/phosphate
buffer solution of pH 8.3 was used in the method. Excitation wavelength of the analyte
was found to be 287 nm while emission was measured at 465 nm. Calibration curve was
linear in the range of 30 – 300 ng/mL. Limit of detection was found to be 10 ng/mL while
limit of quantification was found to be 30 ng/mL. Coefficient of variation of the method
is 2 % for ten replicates. Three different Spanish marketed dosage forms were analyzed
for active moxifloxacin by the method. A modified form of the method is micellar
enhanced fluorometry of moxifloxacin. This medium facilitates the direct determination
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of moxifloxacin in human urine and blood samples by standard addition methods.

Sodium dodecyl sulphate (8 mM) solution in acetic acid-acetate buffer of pH 4.0 was
used in the method. Excitation wavelength of the analyte was found to be 294 nm and
emission was measured at 503 nm. Linearity range of the method was same but with
25 % lower slope while 15 ng/mL and 60 ng/mL were detection and quantification limits
respectively. Precision of the method in terms of coefficient of variation was found to be
1.3 %. The background signals in biological blank samples were minimized in sodium
dodecylsulfate medium and sufficient sensitivity was achieved to measure the
concentration of moxifloxacin in human blood and urine samples after its administration.
Average percent recovery in urine and serum samples was observed to be 102 ± 2 % and
105 ± 2 % respectively.
Lemoine et al. (30) reported a high performance liquid chromatographic method for
determination of moxifloxacin in human lungs tissue and blood samples using UV
detection and Supelcosil ABZ + as analytical column. For liquid-solid extracting, Oasis
column as a stationary phase was adopted in the method. Moxifloxacin was determined in
the range of 0.025 – 3.2 µg/mL in blood samples and 0.25 – 16 µg/mL in lungs tissue
samples. The method can be applied to the pharmacokinetics study of penetration of
moxifloxacin in human lung tissue.
Shao et al. (31) presented a new chemiluminescence method for determination of

levofloxacin. The method is based on quenching effect of levofloxacin in the
chemiluminescence reaction of luminol and myoglobin system. The drop in the
chemiluminescence intensity is proportional to the log of levofloxacin concentration in
the range of 0.07 – 100 ng/mL with correlation coefficient almost one. Limit of detection
was found to be 0.02 ng/mL. Coefficient of variation for five replicates was found ≤ 3 %.
Successful application of the method for assay of the drug in dosage forms and biological
samples was proved. In the application to human urine and serum, no pretreatment of the
samples is required.
Kumar et al. (32) reported a simple, sensitive, fast and selective high performance liquid
chromatographic method for determination of levofloxacin in human blood sample. The
drug was extracted using ethyl acetate. Intensil 5µm C18 column (4.6 x 250 mm) was
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used as stationary phase while mixture of phosphate buffer (pH 2.5) and cyanomethane
(80:20) was used as mobile phase. Flow rate of mobile phase was maintained at 1
mL/min. Plasma matrix showed no interferences with the assay and good resolution of
the chromatogram was obtained. The retention time for levofloxacin was 5.9 ± 0.05 min
while the internal standard was retained for 10.1 ± 0.03 min. Average percent recovery of
the method was found greater than 85 %. Linear range of the method was from 0.1 to 10
µg/mL and correlation coefficient was found to be 0.9994. Reproducibility of the method
was proved with intra and interday precision and accuracy. Pharmacokinetics studies of
the drug were successfully carried out with the method.
Nagavalli et al. (33) reported simultaneous determination of levofloxacin and ornidazole

in a combined product with the help of first order derivative spectrophotometric
technique. Absorbance monitored at 277.5 nm and 319 nm of first order derivative
spectrum were employed for determination of levofloxacin and ornidazole respectively.
The two drugs do not interfere with each other. Beer’s law range was 10 – 50 µg/mL for
levofloxacin and 20 – 80 µg/mL for ornidazole. Accuracy of the method was evaluated
from recovery study and the results were subjected to statistical validation.
Kassab et al. (34) proposed an alternative spectrophotometric analytical method for

determination of levofloxacin in injectable and tablets formulations. Beer’s law was
observed in the range of 3 - 8 µg/mL. Average recovery in tablet was found to be 101.42
± 0.45 % and 100.34 ± 0.85 % in injectable formulations with 1 % coefficient of
variation. Detection and quantification limits were found to be 0.08 µg/mL and 0.25
µg/mL respectively. The proposed method was found applicable to quality control
analysis of the drug.
Fang et al. (35) presented a fast and selective high performance liquid chromatographic –
mass spectrometric method for simultaneous determination of levofloxacin, rifampicin
and isoniazid in mouse plasma and tissue samples. Gatifloxacin was employed as internal
standard. The drugs and internal standard were extracted from plasma and tissue samples
using methanol. Stationary phase of the method was Welch material C4 column and was
operated at 25 oC. Mixture of 0.05 % formic acid and methanol (93:7) was used as mobile
phase. Flow rate was maintained at 1 mL/min. Percent recovery for tissue analysis was in
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the range of 83.3 to 98.8 while for plasma analysis, it was found between 75.5 and 90.8.
Accuracy of the method was 91.7 % to 112.0 % in tissue analysis and 94.6 % to 108.8 %
in plasma analysis. In tissue analysis, less than 13.3% intra and interday precision was
recorded while in plasma analysis, intra and interday precision was found less than 8.2%.
Calibration curve for levofloxacin was linear in the range of 0.13 – 26.2 µg/g in tissues
and 0.09 – 4.53 µg/mL in plasma. Quantification limit of levofloxacin was found to be
0.13 µg/g in tissues while in plasma, it was found to be 21.8 ng/mL. Detection limit was
found to be 0.05 µg/g and 6.6 ng/mL in tissues and plasma respectively. The proposed
method was successfully applied for simultaneous determination of levofloxacin,
rifampicin and isoniazid in different mouse tissues and plasma samples.
Gupta (36) reported a fast, sensitive and selective gradient reversed phase ultra-
performance liquid chromatographic method for determination of levofloxacin. The
method is suitable for determination of levofloxacin in marketed preparations such as
tablets and eye drops and in aqueous humour. Levofloxacin was separated on Waters
Acquity HSS T-3 Column. Run time of the method was as short as 5 minutes.
International Conference on Harmonization’s guidelines were used for validation of the
method in terms of accuracy, precision, linearity, system suitability, detection and
quantification limit, robustness and specificity of the method. Stability of the method was
demonstrated by forced degradation analysis of the drug in bulk. The method was
successfully used for pharmacokinetics study of the drug in the form of eye drops and
analysis of marketed dosages.
Ulu (37) proposed a sensitive spectrofluorimetric method for determination of

levofloxacin, enrofloxacin and ofloxacin in marketed formulations. The method is based
on formation of charge transfer complex between the drugs and chloranil. The reaction
parameters were carefully studied and at optimal parameters, the excitation wavelengths
for the complexes were in the range of 359 – 363 nm and emission wavelengths were in
the range of 442 – 488 nm. Calibration curve was linear in the concentration range of 50
– 1000 ng/mL for levofloxacin, 50 – 1000 ng/mL for enrofloxacin and 25 – 500 ng/mL
for ofloxacin. Limit of detection for levofloxacin, ofloxacin and enrofloxacin was found
to be 17 ng/mL, 8 ng/mL and 17 ng/mL respectively. Interferences of common excipients
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present in marketed formulations were checked and the method was found free from the
effect of these additives. International Conference on Harmonization’s directives were
used in validation of the method, its accuracy, specificity, precision, linearity and
robustness. Marketed formulation can be successfully analyzed with the method. Results
of the proposed method were in good agreement with official method and evaluated
statistically with the help of t- and f-tests.
Shirkhedkar and Surana (38) developed two simple, fast, low cost and accurate
spectrophotometric methods for determination of levofloxacin in bulk and tablets form.
Cyanomethane (10 %) was used as solvent and absorbance of the analyte was measured
at 288 nm. First order derivatization of the same spectrum was done with ultra violet
probe software of the instrument at ∆λ = 4. The changed wavelength for recording
amplitude of the trough was noted 297 nm. Linear calibration curve was drawn for both
methods over the concentration range of levofloxacin 2 – 12 µg/mL with r equal to
0.9999. Results of the methods were in good agreement with labeled value of the drug.
Statistics and recovery results were used for validation of the methods. Precision and
accuracy of the methods were excellent and coefficient of variation was found less than
2 %.
Baietto et al. (39) developed high performance liquid chromatography with UV detection
for simultaneous determination of levofloxacin, linezolide, moxifloxacin and rifampicin
in human blood samples. Organic protein precipitation procedure was adopted in the
technique which ensured simple and fast sample preparation and made the direct injection
of sample to the system possible. With the use of quinoxaline as internal standard, better
precision and accuracy of the method was recorded. The average recovery was found to
be 75.9 % and 0.234 µg/mL was found as limit of quantification for levofloxacin. The
technique was successfully applied for simultaneous determination of the drugs and the
technique was found suitable for the pharmacokinetics study and routine analysis of the
drugs.
Bao et al. (40) reported a sensitive and specific liquid chromatographic mass
spectrometric method for determination of levofloxacin and rifampicin in infected tissues
with in teflon catheter segment which were subcutaneously implanted in mice. Analytes
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wrtr extracted from the tissue using solid phase extraction process while separation and
assay was achieved by reversed phase high performance liquid chromatography coupled
with positive electro spray ionization mass spectrometry. Linearity of the method was
found in the concentration range of 0.02 – 2 µg/g. High accuracy and precision was
perceived from the intra and interday results and -1.3 % relative error was found in
intraday readings while interday percent relative error was calculated as -3.3 %. Less
amount of tissue is required for the assay as compared to the early reported methods and
the proposed method is applicable to determination of fluoroquinolones in a device
related infected tissues.
Zhou et al. (41) reported a high performance liquid chromatographic technique for
determination of levofloxacin in human blood samples. Dichloromethane was employed
for extraction of the drug from sample and phosphate buffer of pH 7 was used for
neutralization of the medium. Terazosin was used as internal standard. Separation of the
internal standard and levofloxacin was achieved by C 18 column and mixture of 10 mM
phosphate buffer (pH 3), cyanomethane and triethylamine (76:24:0.076) was used as
mobile phase, with a flow rate of 1 mL/min. Fluorometric detector was used for
quantification of the analyte using 295 nm and 440 nm as excitation and emission
wavelengths respectively. Calibration curve was plotted in the concentration range of
0.0521 – 5.213 µg/mL. Quantification limit of the technique was found to be 0.521
µg/mL. The retention time for levofloxacin was 2.5 min and 3.1 min for the internal
standard. Precision between the run and within the run was found to be 11 % and 12 %
respectively. The percent relative error of the method for intra and interday was -6.3 and
4.5 respectively. At three different concentrations, recovery was found to be in the range
of 86 – 89 %. The method was found efficient, sensitive and reliable. Bioequivalence and
pharmacokinetics of the drug in healthy volunteers were successfully performed.
Hurtado et al. (42) developed a simple and fast high performance liquid chromatographic
method for determination of levofloxacin. Phenomenex ® C18 column, having 4 µm
particle size, was used as stationary phase while mixture of water, cyanomethane and
0.025 M phosphoric acid (60:20:20) was used as mobile phase (pH 3) with a flow rate of
1 mL/min at room temperature. In high performance liquid chromatographic method,
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detector was set at 294 nm while measurements were performed at 292 nm in UV- visible
method. Levofloxacin was determined by both methods having linear curve in the
specified range and correlation coefficient was found to be greater than 0.999. For
chromatographic method, coefficient of variation was found to be 0.66 % while for
spectrophotometric, it was found to be 1.0 %. The average percent accuracy was 100.68
and 99.61 for chromatographic and spectrophotometric methods respectively. Validation
of the methods was checked in terms of linearity, precision, accuracy, robustness and
specificity of the methods. Determination of levofloxacin in marketed injectable
preparations was done successfully with both the methods.
Kothekar et al. (43) reported an analytical method for determination of levofloxacin and
ambroxol in new tablets formulation. The two drugs were separated using hypersil BDS
C18 column and a mixture of buffer, cyanomethane and methanol (65:25:10) with small
amount of triethylamine as mobile phase while pH was tuned as 5.2 using
orthophosphoric acid. The mobile phase was passed through the column with the flow
rate of 1 mL/min and the run time was found 10 min. The signal was measured at 220
nm. Linear calibration curve was observed in the concentration range of 7 – 22 µg/mL
with correlation coefficient almost equal to 1.0. coefficient of variation was found to be ≤
2 %.
Chepurwar et al. (44) described fast, simple and accurate high performance thin layer
chromatographic method for simultaneous determination of levofloxacin and ornidazole
in tablets form. With Merck aluminum sheet of silica gel and n-butanol, methanol and
ammonia (5:1:1.5) as mobile phase, separation of the drugs was achieved and 298 nm
was used for densitometric study of their dots. The method was rectilinear for
levofloxacin in the range of 50 – 250 ng/spot. Interferences study of common additive
found in marketed formulations was performed and the method was found fit for the
analysis of dosage form. International Conference on Harmonization’s guidelines were
followed for validation of the method and the method was suitable for quantitative
analysis of the drugs.
Gao et al. (45) reported fast and simple high performance liquid chromatographic method
for determination of levofloxacin in plasma samples. Kromasil C 18 column and mixture of
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water, cyanomethane, phosphoric acid and triethylamine (86:14:0.6:0.3) as mobile phase

and UV detector at 294 nm were used for analytical separation. The flow rate was
maintained at 1 mL/min. Signal response versus concentration was linear in the range of
0.05 – 5.0 µg/mL with correlation coefficient greater than 0.99. The technique was
accurate, precise and was fit for routine bioequivalence study. Bioequivalence analysis of
two branded levofloxacin in healthy Chinese volunteers was successfully carried out with
the proposed method.
Askal et al. (46) reported a spectrophotometric method for determination of

fluoroquinolones. The fluoroquinolones were reacted with excess of N-
bromosuccinimide and converting the excess reagent to a violet colour product with p-
phenylenediamine. The product showed maximum absorbance at 530 nm. Reaction
conditions and reagent concentration were carefully investigated and optimized. NMR,
UV-vis and IR techniques were used to propose mechanism of the reaction and molar
ratio of the reagents was determined. Linearity of the method was in the general
concentration range of 3 – 25 µg/mL. Limit of detection of the method was in the range
of 0.33 – 1.29 µg/mL while quantification limit was found in the range 1.10 – 4.31
µg/mL. Coefficient of variation for all fluoroquinolones did not go beyond 2% and
precise results were obtained. The investigated drugs were successfully analyzed in their
pure and marketed preparations and the additives posed no interferences with the method
except ascorbic acid. The interferences due to ascorbic acid were eliminated by its
reaction with KBrO3 prior to analysis. Percent accuracy of the method was found in the
range of 99.85 % – 100.17 % ± 0.13 – 0.59. Good agreement was observed between the
results of the proposed method and official and literature method.
Salem et al. (47) reported a simple, sensitive, selective and accurate atomic absorption
spectrometric method for determination of levofloxacin and nine other fluoroquinolones.
Precipitation reaction between the fluoroquinolones and iron (III) chloride, Copper
acetate and silver nitrate was exploited in the method. Conditions for high yield of
precipitates and its solubility were optimized. Ions were directly determined in precipitate
or indirect in the filtrate by atomic absorption spectrometery. Calibration curve was
linear for each of the studied drug over the range of 10 – 100 ng/mL. The limit of
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detection and quantification was in the range of 1.125 to 2.754 ng/mL and 3.425 to 5.986
ng/mL respectively. Job’s method was employed for determination of molar composition
of the complexes and association constants were worked out. The selected
fluoroquinolones were successfully determined in their pharmaceutical formulations by
the proposed method. The results were compared with literature and official methods and
good agreement was found in accuracy and precision supported by statistical parameters.
The proposed method was also applied to the assay of the drugs in spiked samples of
biological nature.
Salem et al. (48) developed a highly sensitive spectrofluorimetric method for

determination of ten fluoroquinolones antibacterial in their marketed formulations and
biological samples. The fluoroquinolones were levofloxacin, ofloxacin, enrofloxacin,
amifloxacin, difloxacin, ciprofloxacin, pefloxacin, lomefloxacin, norfloxacin and
enoxacin. The method is based on charge transfer complex formation between the drugs
and bromanil. Formation of the complex was confirmed by IR and UV studies. These
complexes were pretty stable and enhanced the fluorescence intensity many folds.
Optimization study of reaction parameters was carried out. At optimal conditions
excitation wavelengths of the complexes were in the range of 275 – 290 nm and emission
was measured in the range of 450 – 470 nm. Calibration curve was linear in the
concentration range of 0.02 – 3.1 µg/mL for all the drugs. Application of the method to
marketed formulations was checked. The results were compared with reference and
official methods and good precision and accuracy was observed and verified by t- and F-
tests. The method was applied for determination of the drugs in samples of human urine.
Sun et al. (49) described a novel, fast and sensitive chemiluminescence method for
determination of levofloxacin and ofloxacin. Mixture of Ce (IV), sodium dithionite, the
drug and sulphuric acid was used as chemiluminescence system. Optimum conditions for
the process were explored and mechanism was proposed for enhanced
chemiluminescence. Linearity of the method was in the concentration range of 1.0 x 10 -7
– 6.0 x 10-6 g/mL and 1.0 x 10-8 – 1.0 x 10-7 g/mL for levofloxacin and ofloxacin
respectively. Correlation coefficient for the curve was found to be 0.9988. Limit of
detection was found to be 7 x 10 -9 g/mL. Coefficient of variation for eleven replicates
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was found to be 2 % for levofloxacin at 4 x 10 -7 g/mL while for ofloxacin at 6 x 10-7

g/mL. The method was applied to the determination of the drugs in marketed
formulations and samples of biological interests and satisfactory results were obtained.
Shao et al. (50) reported a new flow injection method for quantification of levofloxacin.
The method is based on enhanced chemiluminescence intensity by Luminol-KIO 4 system.
The relation between enhanced intensity and concentration of levofloxacin was linear in
the range of 7 – 1000 ng/mL. Limit of detection of the method was found to be 2 ng/mL.
Maintaining 2 mL/min as the flow rate, analyses could be completed just in 30 seconds.
Coefficient of variation for five replicates was about 3 %. The percent recovery of the
drug was found in the range of 91.0 % – 109.4 %. The quantification of levofloxacin in
dosage tablets, eye drops and human urine and serum samples was successfully achieved
with the method.
Santro et al. (51) developed an analytical chromatographic method for the determination
of third generation fluoroquinolones in injectable and tablet formulations. Levofloxacin,
lomefloxacin, gatifloxacin and pefloxacin were analyzed on the method at room
temperature. The method is simple and fast. Stationary phase of the method was
LiChrospher® RP 18 column while mobile phase was mixture of water and cyanomethane
(80:20) containing a small amount (0.3 %) of triethylamine. Phosphoric acid was used to
adjust pH at 3.3. The analytes were monitored in the wavelengths range of 279 nm to 295
nm. Time required for the method was 5 minutes. Linear calibration curves were drawn
in the concentration range of 4.0 – 24.0 µg/mL with 0.9999 as correlation coefficient.
The average recovery was greater than 99.54 % and coefficient of variation was found
less than 1 %.
Siewert (52) reported high performance liquid chromatographic method for the routine
analysis of levofloxacin in blood samples and dialysate. Sample preparation was made
simple by single step protein precipitation from plasma while dialysate was directly
injected to the system. YMC Pro C18 RP column was used for separation of the analyte.
Fluorescence detection system was used in the method and excitation wavelength was
found to be 296 nm while emission was measured at 504 nm. The emission intensity
versus concentration was linear in the concentration range of 0.1 – 6.0 µg/mL for plasma
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and 0.1 – 5.0 µg/mL for dialysate. Percent relative error in the intera and interday
accuracy and precision was found less than 10 %. Degradation products of levofloxacin
were not found in both matrices. Pharmacokinetic of the drug in vitro and real samples
analysis were successfully performed with the proposed method.
Geffken and Salem (53) developed a highly sensitive spectrofluorimetric method for
determination of ten fluoroquinolones in their marketed formulation and biological
samples. The fluoroquinolones were levofloxacin, ofloxacin, enrofloxacin, amifloxacin,
difloxacin, ciprofloxacin, pefloxacin, lomefloxacin, norfloxacin and enoxacin. The
method is based on charge transfer complex formation between the drugs and fluoranil.
IR and UV studies confirmed formation of the charge transfer complex. These complexes
were pretty stable and enhanced the fluorescence intensity many folds. At optimal
conditions excitation and emission wavelengths of the complexes were in the range of
270 – 285 nm and 450 – 460 nm respectively. Fluorescence Intensity versus
concentration was linear in the concentration range of 0.02 – 3.1 µg/mL for all the drugs.
Application of the method to marketed formulations was carried out. The results were
compared with reference and official methods and good precision and accuracy was
observed and evaluated by t- and F-tests. The method was applied for the determination
of the drugs in samples of human urine.
Salem (54) described three methods for the determination of levofloxacin, ofloxacin,
ciprofloxacin, pefloxacin, enrofloxacin, amifloxacin, lomefloxacin, norfloxacin,
difloxacin and enoxacin in pharmaceutical preparations and biological samples. The first
method is based on direct fluorometry of the drugs in 0.1 N sulphuric acid. All these
drugs were native fluorescent in the given medium and excitation wavelength was noted
290 nm and emission wavelength was found 450 nm. The signal versus concentration
was linear in the concentration range of 0.3 – 1.4 µg/mL. The second method is based on
atomic absorptivity. The drugs were reacted with cobalt sulphate to give an insoluble
product. Optimum pH value and ionic concentration of buffer for maximum precipitation
was investigated. Direct and indirect estimation of the metal in the precipitate was carried
out using atomic absorption. Linear calibration curves were drawn in the range of 3 – 30
µg/mL for all the mentioned drugs. Job’s method was used for determination of molar
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ratio of the complex and their formation constant was calculated. The third method is
spectrophotometric and is based on the oxidation of the drugs by ammonium vanadate in
the presence of sulphuric acid. Vanadium (V) is reduced to Vanadium (IV) which has a
characteristic greenish colour which was monitored at 766 nm. Reaction parameters such
as reagent concentration, heating temperature and time and concentration of sulphuric
acid were carefully optimized. Beer’s law was observed in the concentration range of 10
– 40 µg/mL and evaluation of precision and accuracy of the method was done by
replicate analysis in the given range. Correlation coefficient found was almost 0.9994 for
all the drugs. All the methods were tried for quantification of the investigated drugs in
marketed dosage forms and the results were compared with reference and official
methods. Statistical parameters such as t- and F-tests were applied and good precision
and accuracy was observed. Fluorometric method was found the most sensitive among
the three methods. The other two methods were applied to quantification of the drugs in
spiked plasma and urine samples. The methods were found simple, sensitive, accurate
and selective.
Ashour and Al-Khalil (55) reported spectrophotometric methods for determination of

levofloxacin in its pure and dosage forms. Coloured ion-pair complexes of the drugs were
produced with bromocresol green (BCG) and bromophenol blue (BPB) in acidic aqueous
medium. The complexes are extractable in trichloromethan and LEV-BCG has maximum
absorbance at 428 nm while LEV-BPB showed maximum absorbance at 424 nm.
Absorbance versus concentration curve was linear over the drug concentration range of
1.85 – 25 µg/mL for method I (LEV-BCG) and 1.85 – 31.5 µg/mL for method II (LEV-
BPB). The method is free from the effect of excipients present in the dosage forms.
Marketed forms of the drug were analyzed successfully and the results of the methods
were supported by statistical operations.
Du et al. (56) reported a very sensitive spectrofluorimetric method for determination of

levofloxacin, pefloxacin, norfloxacin and lomefloxacin in their marketed dosage forms
and biological samples. The method is based on formation of charge transfer complex
between the drugs and tetracyanoethylene. The charge transfer complex was found stable
and has greater fluorescence intensity than either of the drugs. Infra-red spectroscopy was
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used for the confirmation of complexes. Optimum reaction conditions for the formation
of the complex were investigated. Under optimized conditions, linear calibration curve
was plotted in the concentration range of 2.0 – 900 ng/mL. Limit of detection for
levofloxacin, pefloxacin, norfloxacin and lomefloxacin were found to be 0.4, 0.6, 1.0 and
0.8 ng/mL respectively. Good accuracy and precision was observed when the method was
applied to quantification of the drugs in dosage forms. Application of the method to urine
samples was also carried out.
Du et al. (57) reported a spectrofluorimetric technique for determination of levofloxacin,

ofloxacin, lomefloxacin and pipemidic acid. The method is based on formation of charge
transfer complex between the drugs and tetracyanoquinodimethan. The complex was
found stable and having 15 – 90 times high fluorescence intensity than the drugs itself.
The formation of complex was verified using UV-Vis and IR spectroscopy. Optimum
conditions for the formation of the complex were investigated and linear calibration curve
was constructed in the concentration range of 0.02 – 2.2 µg/mL for all the drugs.
Detection limit of the method was in the range of 0.006 – 0.016 µg/mL. Application of
the proposed method for quantification of the investigated drugs was compared with
official and reference methods and precision and accuracy of the method was evaluated
from t- and F-tests. Spiked biological samples were analyzed with the method too.
El-Brashy et al. (58) developed two simple, sensitive and fast spectrophotometric
methods for levofloxacin, ciprofloxacin and norfloxacin. In both methods, coloured
binary complexes were produced between the drugs and merbromin and eosin Y in
buffered aqueous medium. Optimum conditions for the analysis were explored and at
optimum conditions, complex with merbromin showed maximum absorption at 545 nm
and complex with eosin Y exhibited maximum absorption at 547 nm. Using the first dye,
the response of the signal versus concentration was linear in the range of 2 – 15 µg/mL
while for the second dye, it was found linear in the range of 2 – 8 µg/mL. Average
recoveries with merbromin dye was found to be 99.960 ± 0.491 for levofloxacin, 99.980
± 0.506 for ciprofloxacin and 100.017 ± 0.510 for norfloxacin while with eosin Y, 99.935
± 0.648 for levofloxacin, 100.01 ± 0.606 for ciprofloxacin and 99.973 ± 0.678 for
norfloxacin. The drugs in their marketed forms and spiked samples of human urine were
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analyzed by the proposed method and the results were in good agreement with official
methods. No solvent extraction of the complexes was required in the proposed methods
and this was one of the advantages of the methods. The methods were found economical,
specific and speedy and therefore, could be adopted for routine quantification of the
drugs in quality control labs.
El-Brashy et al. (59) reported two spectrophotometric techniques for determination of

levofloxacin, ciprofloxacin and norfloxacin in their pure and marketed formulations. In
method I, a yellow coloured complex was formed between the drugs and bromocresol
green. The complexes of levofloxacin and norfloxacin were directly prepared in
dichloromethane while the complex with ciprofloxacin was prepared in aqueous acidic
buffer and was extracted to dichloromethane. The drugs were indirectly determined by
measuring absorbance of the analyte at 411 nm and 412 nm respectively. Absorbance was
proportional to the drug concentration in the range of 1 – 20 µg/mL while average
percent recovery for levofloxacin was found to be 100.41 ± 0.72, 100.23 ± 0.9 for
ciprofloxacin and 99.99 ± 0.54 for norfloxacin. Method II is based on formation of
charge transfer complex of levofloxacin with p-chloranilic acid and norfloxacin with
tetracyanoethylene. Cyanomethane was used as solvent. The first complex absorbs
maximum at 521 nm while the second was measured at 333 nm. Detector response versus
concentration of the drugs was linear in the concentration range of 15 – 250 µg/mL for
levofloxacin and average percent recovery was found to be 99.89 ± 0.45. The proposed
methods were applied for determination of the drugs in marketed forms and the results
were found in good agreement with reference assays. The proposed methods can be
adopted for routine determination and quality control labs.
Djabarouti et al. (60) reported a specific and sensitive high performance liquid
chromatographic method for determination of levofloxacin in biological samples such as
bone tissue, human plasma and bronchoalveolar lavage. The instrument was equipped
with fully automatic solid phase extraction for the samples using OSAIS cartridge.
Supelcosil ABZ+ column was employed for separation of the analyte with UV detection
at 299 nm. The method response was linear in the concentration range of 0.5 – 10 µg/mL
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in bone tissues, 0.25 – 25 µg/mL for the drug in plasma and 1- 6 µg/mL in
bronchoalveolar lavage.
Altiokka et al. (61) reported three methods for determination of levofloxacin. The first
method is flow injection analysis having UV detection systems, while the second is
potentiometric and the third one is based on conductometry. Mixture of acetate buffer
(0.2 M) of pH 3 and methanol (90:10) was used as solvent in all the three methods. The
pump in flow injection method was set at the rate of 1 mL/min. Limit of detection and
quantification were calculated at λmax (288 nm). Marketed formulations were analyzed by
the methods and coefficient of variation was found to be 0.83 for flow injection analysis,
0.98 for potentiometric and 0.99 for conductometric method.
González et al. (62) proposed a spectrofluorimetric method for determination of

levofloxacin in pharmaceutical formulations and spiked biological samples. The method
is based on direct fluorometry of aqueous solution of the drug in acetate buffer (pH 4).
Wavelengths of excitation and emission used were 292 nm and 494 nm respectively.
Linear calibration curve was drawn in the concentration range of 20 – 3000 ng/mL.
Sensitivity of the method was improved by the use of 8 mM sodium dodecyl sulphate
solution at pH 5 and quantification of levofloxacin in spiked samples of human urine and
serum was made possible.
Sharma and Sharma (63) reported three different spectrophotometric methods for
determination of sparfloxacin in tablet forms. Method A is based on direct
spectrophotometry of sparfloxacin in basic methanolic medium. Maximum absorption
was observed at 280 nm. Method B is first order derivative spectroscopic technique. A
sharp peak at 267 nm in the first order derivative spectra was observed and selected for
the assay. Method C is area under curve method and was operated in the wavelength
range of 296 – 298 nm. Calibration curve was drawn for all the three methods in the
concentration range of 5 – 40 µg/mL. These methods were found sensitive, fast, accurate
and selective and were applied for quantification of sparfloxacin in dosage tablet form.
Recovery tests of the methods were good and the results were statistically verified.
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Noh et al. (64) reported a mass spectrometric method for determination of sparfloxacin in
rat plasma. The assay was carried out after a single oral administration of the drug to rats.
Plasma was deproteinated using cyanomethane. A reversed phase HPLC having C 18
column and tandem mass spectrometry with electrospray ionization as detection tool
were used for the analyte in the method. The method is in good agreement with Food and
Drug Administration guidelines.
Adegoke and Balogun (65) reported a simple, sensitive and accurate spectrophotometric
method for determination of sparfloxacin, ciprofloxacin and pefloxacin. Excess of Ce
(IV) in the presence of perchloric acid was used for oxidation of the drugs and surplus Ce
(IV) was reacted with para dimethylaminobenzaldehyde to yield a brown yellow product.
The product showed λmax at 470 nm. Drug concentration in the test solution was
correlated with decrease in absorption of the coloured product. Reaction parameters were
carefully studied and optimal temperature 30 oC and optimal heating time 5 minutes were
selected. The absorption signal was linear in the concentration range of 1.5706 – 6.2824
µg/mL for ciprofloxacin, 15.8 – 47.4 µg/mL for pefloxacin and 1.86 – 7.44 µg/mL for
sparfloxacin with good correlation coefficient of 0.9996, 1.000 and 0.9977 respectively.
Coefficient of variation for the method was found to be 2 %. The method was found
better in terms of speed, sensitivity, precision and accuracy when results of the method
were compared with a reference non-aqueous titration method. Validation of the method
was carried out in terms of accuracy, ruggedness and precision.
Kamlesh et al. (66) reported two spectrophotometric methods for determination of

sparfloxacin in bulk and marketed formulations. Method one is based on diazotization of
the drug, yielding a coloured product which was measured at 549 nm. The second method
is based on oxidation of the drug using iron (III) and subsequent reaction of iron (II) with
1,10-phenonthroline to form a red colour complex which was measured at 465 nm. These
methods were applied for the analysis of tablet dosage forms and results comparable with
the labeled value were obtained.
El-Didamony (67) reported a simple and sensitive spectrofluorimetric method for

determination of sparfloxacin. The method was developed using Yttrium sensitized
fluorescence. Optimizations of experimental conditions were carried out and a buffer
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solution of pH 8.0 was used during the assay. Linearity of the method was in the
concentration range of 8 x 10-7 to 1.4 x 10-4 mol/L of sparfloxacin with good (0.9997)
correlation coefficient. Limit of detection was found to be 9.01 x 10 -8 mol/L. The
proposed mechanism of the sensitization was established. Applicability of the method to
dosage forms and human samples of urine and serum was checked and satisfactory
results were obtained.
Srikar et al. (68) reported two methods for determination of sparfloxacin in bulk and
marketed preparations. The first method is based on reaction of sparfloxacin with para
dimethyl amino cinnamaldehyde to yield Schiff’s base which absorbs at 465 nm. The
Beer’s law range was 10 – 50 µg/mL, regression equation was Y = 0.0193X – 0.0038 and
average percent recovery was 99.73. In the second method, the drug was oxidized by iron
(III) chloride followed by complexation of iron (II) with 1, 10-phenonthroline having λ max
515 nm. The Beer’s law range was 5 – 25 µg/mL, regression equation was Y = 0.0245X
– 0.0124 and average recovery was 99.13 %. Validation of both methods was carried out
and satisfactory results were obtained.
Wang et al. (69) reported three analytical methods for determination of six different
fluoroquinolones. These include ciprofloxacin, norfloxacin, sparfloxacin, lomefloxacin,
levofloxacin and ofloxacin. Britton-Robinson buffer in the pH range of 4.2 – 5.0 was
used in all the three methods. The basic chemical reaction involved in the methods is the
formation of chelate cation of the drugs with Cu (II) ion. The reaction of the chelate
cation and erythrosine results in the formation of ion association complex. The ion
association complex has different absorption wavelength than the drugs and reagents. In
spectrophotometric method, the decolorization of erythrosine at 526 was monitored for
determination of the drugs. The formation of ion association complex resulted in
quenching of fluorescence, and this property was exploited in assay of the drugs using
spectrofluorimetry. These both methods were quite sensitive and limit of detection for the
drugs was in the range of 7.1 – 12.2 µg/mL. In the third method, enhancement of
resonance Rayleigh scattering, due to the above chemical reaction, was used for
determination of the drugs. All the complexes gave the scattering peak at 566 nm. Two
other minor peaks were also observed, one at 333 nm and the other at 287 nm. In
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resonance Rayleigh scattering method, the selected drugs could be detected in the range
of 1.70 – 3.10 µg/mL. In all the three methods, the last one was more sensitive.
Experimental conditions for all the methods were carefully studied and density function
theory and AM1 method were used to establish the mechanism of the reaction.
Rahman and Ahmad (70) reported a simple, fast and sensitive high performance liquid
chromatographic method for determination of sparfloxacin. Cyanomethane (35%) in
buffer system (sodium di hydrogen phosphate 40 mM in deionized water) was used as
mobile phase in the method. Stock solution (0.05 mg/mL) of sparfloxacin was prepared
using the buffer system as solvent. UV-spectrophotometer was used to find λ max (292 nm)
for sparfloxacin solution. Four different brands of marketed pills were successfully
analyzed with the method and the results were found acceptable and reproducible.
Reddy et al. (71) reported an electrochemical method for determination of sparfloxacin.

The method is based on the electrochemical activity of the drug on β-cyclodextrin
modified carbon paste electrode. The proposed method was compared with carbon paste
electrode and a major enhancement was found in current signal of the drug. Cyclic
voltammetric investigation showed that the process was adsorption controlled.
Du et al. (72) reported an indirect spectrofluorimetric method for determination of

sparfloxacin tablets. Sparfloxacin was oxidized by nitrous acid followed by its reaction
with chlorohydric acid to obtain chloro-sparfloxacin. Molecular structure of the
derivative was established with elemental analysis, IR and MS. Fluorescence activity of
sparfloxacin and its derivative was quite different. Sparfloxacin had almost no fluorescent
activity and could not be directly determined spectrofluorimetrically but the derivative
had a distinct peak in the emission spectrum. Sparfloxacin had 0.0072 as fluorescence
quantum production rate while the derivative has 0.1539. Percent recovery of the method
was found to be 97.2 % with coefficient of variation of 1.6. The proposed mechanism of
the reaction was established.
Kuchekar et al. (73) reported two methods for quantification of sparfloxacin in dosage
tablets. In one of the methods, iron (III) was reduced by the drug to iron (II) and then
reacted with potassium dichromate to yield a green colour product which was monitored
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at 680 nm against the blank. The method is linear over the concentration range of 1.5 –
5.5 µg/mL. In second method, a similar mechanism was followed using iron (III) nitrate
and potassium ferricyanide to give a blue colour product which was measured at 720 nm
against a reagent blank. Beer’s law was obeyed in the range of 1.5 – 4.0 µg/mL.
Sankar et al. (74) described a simple spectrophotometric method for the determination of
sparfloxacin. The diazotized drug was coupled with phloroglucinol to yield a light green
product which was measured at 430 nm.
Marona et al. (75) compared high performance liquid chromatography, microbiological

bioassay and UV spectrophotometric methods for determination of sparfloxacin in dosage
pills. Each method was found satisfactory in terms of accuracy, precision and
reproducibility. The results of each method were reliable with in specified range. The
specific among these methods were high performance liquid chromatography and
microbiological bioassay but high performance liquid chromatography has high running
cost and microbiological bioassay is time consuming. Adoptability of the methods for
routine analysis of the drug was checked in terms of running cost, instrumentation,
simplicity, speed, chemical reagents and suitability for heavy and light duty.
Marona and Schapoval (76) described a sensitive and accurate spectrophotometric

method for determination of sparfloxacin in dosage tablets. The method is based on
formation of ion-association complex between bromothymol blue (0.5 %) and the drug.
The complex was yellow in colour and showed maximum absorption at 385 nm.
Absorption versus concentration curve was linear in the range of 2 – 12 µg/mL. The
method was found free of interference of common excipients present in marketed dosage
tablets.
Cao et al. (77) reported high performance liquid chromatographic method for
determination of sparfloxacin in injectable formulations. Analytical column, Waters
Symmetry C18, maintained at 30 oC, while UV detector, set at 298.8 nm were used in the
method. Mixture of potassium dihydrogen phosphate (0.2 %), cyanomethane and
methanol (80:15:5) was used as mobile phase with a flow rate of 1.0 mL/min. The
method was linear in the concentration range of 39.94 mg/L to 199.68 mg/L with
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correlation coefficient of 0.9999. The average percent recovery for five analyses was
found to be 100.1 % with relative standard deviation of 0.72 %. Coefficient of variations
for intra and interday analyses of the drug were calculated as 0.14 % and 0.13 %
respectively. Keeping in view the results, the method was found accurate, precise, fast,
sensitive and stable.
Ming et al. (78) reported a new reversed phase high performance liquid chromatographic
method for the determination of sparfloxacin in human urine sample. The method is a pre
column derivatization method and sparfloxacin was first oxidized by nitrous acid and
then reacted with hydroiodic acid to give a fluorophore. The linear range for the method
was found to be 0.05 – 4.0 mg/L. Percent recoveries of the method was in the range of
91.5 – 95.7 and relative standard deviation was found in the range of 1.2 – 4.2 %. The
method was found suitable for determination of sparfloxacin in human urine sample.
Rizk et al. (79) reported a very sensitive method for determination of sparfloxacin,
enrofloxacin, oxolonic acid and flumequine in their dosage forms and liquids of
biological nature. Native fluorescent character of sparfloxacin in cyanomethane and
enhancement in fluorescence intensity due to the interaction between the drugs and
aluminum chloride was exploited in the method. pH of the medium for sparfloxacin was
8 – 8.5, for oxolonic acid was 5 – 6 and for flumequine and enrofloxacin, it was 3.5. The
effects of various experimental parameters were carefully investigated and analyses were
carried out at optimum conditions.
Liming et al. (80) reported a sensitive fluorimetric method based on forming a derivative
of sparfloxacin by oxidizing the drug by nitrous acid followed by reacting with
hydrobromic acid. The derivative was highly fluorescent. The fluorescent character of the
derivative was exploited and an indirect method for determination of sparfloxacin in
human urine sample was proposed. Satisfactory results were obtained.
Du et al. (81) reported indirect spectrofluorimetric method for determination of

sparfloxacin. The drug was oxidized by nitrous acid followed by its reaction with
potassium iodide in acidic medium. The product obtained had reasonable fluorescence
activity and was used for indirect determination of the drug in human urine sample.
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Excitation wavelength of the derivative was 286 nm and emission was observed at 464
nm. The fluorescent intensity of the derivative was 134 fold greater than sparfloxacin.
Fluorescence intensity versus concentration of the drug was linear in the range of 2 x 10 -8
– 1 x 10-6 mol/L. Sparfloxacin can be detected at concentration level of 1.5 x 10 -10 mol/L
by the proposed method. Reaction mechanism was proposed and fluorescence behavior
of the derivative was discussed.
Kumar et al. (82) reported two simple and sensitive spectrophotometric methods for
determination of sparfloxacin in bulk and marketed formulations. The first method is
based on the direct determination of sparfloxacin in methanolic medium and absorption
was measured at 295.2 nm. In the second method the drug was diazotized and then
coupled with resorcinol in basic medium. The resulting dye showed maximum
absorbance at 350 nm which was used for indirect determination of sparfloxacin.
Statistical validation of the method showed that the method is accurate and precise.
Madhuri et al. (83) reported a simple, fast and sensitive ultra violet spectrometric method
for determination of gatifloxacin in raw materials and marketed formulations.
Complexation of the drug was performed with Tween 20, a neutral surfactant. pH of the
medium was maintained at 7.4 using phosphate buffer. The analyte showed maximum
absorption at 295 nm and Beer’s law was obeyed in the concentration range of 2 – 14
µg/mL. Detection and quantification limits were calculated and coefficient of variation of
the method was found less than 1.242. The recovery results were in good agreement with
labeled values.
Patel et al. (84) reported a simple, precise and accurate reversed phase high performance
liquid chromatographic method with UV detection (320 nm) for simultaneous
determination of gatifloxacin and satranidazole in the combined dosage form. Analytes
were separated using Lichrospher 100 C18 column as stationary phase and mixture of
water, cyanomethane and triethylamine (75:25:0.35) was used as mobile phase. O-
phosphoric acid (10 % v/v) was used for pH adjustment of the medium to 3.2 ± 0.02.
Flow rate of the mobile phase was maintained at 1 mL/min and 20 µL of the sample was
injected each time. The retention time for gatifloxacin and satranidazole was found to be
3.44 and 6.0 minutes respectively. Validation of the method in terms of precision,
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accuracy, linearity, specificity and robustness was performed. Linear range for both drugs
was 1 – 70 µg/mL. Recovery studies showed 100.20 % and 99.80 % recovery for
gatifloxacin and satranidazole respectively. Detection and quantification limit of
gatifloxacin were found to be 0.5 µg/mL and 1.0 µg/mL respectively. The method was
found suitable for determination of the drugs in bulk and therapeutic dosage.
Srinivas et al. (85) developed selective, accurate and sensitive high performance liquid
chromatographic method for determination of gatifloxacin, moxifloxacin and
sparfloxacin in plasma sample. Levofloxacin was used as internal standard. Ethyl acetate
was used for extraction of the drugs from plasma using simple liquid-liquid extraction
procedure. A mixture of phosphate buffer (pH 2.5) and cyanomethane (80:20) was used
as mobile phase. The time required for completion of the method was 18.0 min while
gatifloxacin was eluted after 10.8 min. The elution time for internal standard,
sparfloxacin and moxifloxacin was found 6.0 min, 12.8 min and 17.0 min respectively.
The method is applicable in the concentration range of 100 – 10000 ng/mL with ≥0.999
as correlation coefficient. Limit of quantification for the three fluoroquinolones was 100
ng/mL. Food and Drug Administration guidelines were followed in the intra and interday
evaluation of the method and the results were found in the given range. Pharmacokinetics
study of the gatifloxacin in human volunteers was carried out using the proposed method.
Amin et al. (86) reported simple and fast spectrophotometric methods for determination
of gatifloxacin in raw material and dosage formulations. These methods are based on
formation of ion association complexes of the drug with sulphonphthalein acid dyes. The
dyes used were bromocresol green, bromophenol blue, bromocresol purple and
bromothymol blue. Acidic medium in the pH range of 3.0 – 3.4 was maintained with
phthalate buffer. In all the methods, a yellow coloured complex was produced at once
which was extractable to trichloromethan. Maximum absorption was noted in the
wavelength range of 412 – 417 nm. The complexes were stable and no evident change in
absorbance was observed in 48 hours. Beer’s law range was 2.0 – 20.0 µg/mL for
bromocresol green , 2.0 – 14 µg/mL for bromocresol purple and 2.0 – 16 µg/mL for
bromophenol blue and bromothymol blue. Job’s method was used for determination of
composition of the complexes and was 1:1 in all the cases. Successful application of the
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method was carried out for determination of gatifloxacin in bulk and marketed
formulations. Good agreement in results of the proposed method and official methods
was observed.
Sultana et al. (87) reported a simple and specific reversed phase high performance liquid
chromatographic technique using C18 Meditrerranea column and UV detector for
determination of gatifloxacin in bulk, marketed formulation and human blood samples.
Mixture of methanol, cyanomethane and water (2:2:1) was used as mobile phase with a
flow rate of 1 mL/min. pH of the medium was adjusted to 2.7 using phosphoric acid. The
method is free of interferences of the common additives present in marketed preparations
and metal traces. Percent accuracy of the method was found in the range of 99.18 –
191.87 %. Interday variation in the range of 0.32 – 1.80 % and intraday variation in the
range of 0.14 – 1.67 % proved precision of the method. The signal versus concentration
was proportional in the concentration range of 0.1 – 25 µg/mL with 0.999 as correlation
coefficient. Limit of detection and limit of quantification was found to be 1.73 ng/mL and
5.77 ng/mL respectively. Application of the method in routine analysis of the gatifloxacin
in dosage formulation and plasma samples of human blood was successively carried out.
Ilango et al. (88) reported two simple and sensitive UV-visible spectroscopic methods for
determination gatifloxacin in marketed formulations. The first method is based on direct
spectrometry of methanolic solution of the drug. The absorption of the drug was
monitored at 295 nm. In second method, a yellow colour was obtained by the reaction of
the drug with 3-methyl-2-benzthiazolinone (0.2 % w/v) in the presence of iron (III)
chloride (1 % w/v). The derivative showed maximum absorbance at 433 nm. The Beer’s
law range for method one was found to be 2 – 10 µg/mL while for method two, the range
was 50 – 150 µg/mL. The methods were found accurate, precise and reproducible and
could be applied successfully for quantification of gatifloxacin in dosage tablets.
Salgado et al. (89) reported a specific, precise and sensitive high performance liquid
chromatographic method for determination of gatifloxacin in bulk and dosage tablets.
Reversed phase chromatography was carried out on Absorbosphere C18 column using
mixture of 5 % acetic acid, cyanomethane and methanol (70:15:15) as mobile phase with
a flow rate of 1.0 mL/min using isocratic pump. Linearity of the method was found in the
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concentration range of 4.0 – 14.0 µg/mL. Precision of the method in respect of interday
and intraday analysis was checked and good results were obtained with coefficient of
variation less than 1.05 %. The method is free of the interferences of the common
excipients found in tablets formulations. Validation in terms of linearity range, accuracy,
precision, recovery and specificity produced good results.
Suhagia et al. (90) reported a simple and sensitive chromatographic method based on thin
layer technique for determination of gatifloxacin and ornidazole in its combined dose.
The analytes were separated by silica gel 60 F 254 thin layer plates and mixture of n-
butanol, ammonia (6 M) and methanol (8:1.5:1) as mobile phase. Camag TLC scanner 3
was used for scanning the plates at 302 nm. R f value of gatifloxacin was found to be 0.21
± 0.02 while for ornidazole, it was 0.76 ± 0.04. Linear range for gatifloxacin was 100 –
500 ng/spot while for ornidazole, the linear range was 250 – 1250 ng/spot. Limit of
detection of gatifloxacin and ornidazole were found to be 40 ng/spot and 100 ng/spot
respectively. A combined dosage formula of the two drugs was analyzed by the proposed
method.
Ocana et al. (91) reported a spectrofluorimetric method for determination of gatifloxacin

in spiked biological samples. Fluorescence activity of gatifloxacin in aqueous medium
containing citrate buffer of pH 3.5 was explored and 292 nm was found as excitation
wavelength and 484 nm as emission wavelength. The fluorescence intensity versus
concentration of the drug was linear in the range of 0.04 – 0.700 µg/mL. Sodium dodecyl
sulphate (12 mM) was used for enhancement of the fluorescence intensity almost 75 % of
the drug with slight shift in emission wavelength to 470 nm and the calibration curve was
linear 0.020 – 0.450 µg/mL. Spiked human samples of urine and serum were successfully
analyzed by the two methods.
Colunga-Gonzalez et al. (92) developed a fast and simple fluorometric method for
determination of gatifloxacin in semen samples. Excitation wavelength of the analyte
molecules was found to be 300 nm and emission was measured at 486 nm. pH of the
medium was maintained at 3.0. Limit of detection and limit of quantification were found
to be 0.06 µg/mL and 0.2 µg/mL respectively. Coefficient of variation was found to be
1.3 % which indicated precision of the method. The range of percent recovery was found
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62 – 72 %. The method was successfully applied for the determination of gatifloxacin in

semen sample of patients under treatment for genital tract infections.
Venugopal and Saha (93) reported new, simple and economical spectrophotometric
methods for determination of gatifloxacin in raw and marketed products. These methods
are based on direct spectrometry of the drug in different medium. In one of the methods,
100 mM phosphate buffer of pH 7.4 was used and absorption was recorded at 286 nm
while in second 100 mM hydrochloric acid at pH 1.2 was used and absorption was
measured at 292 nm. Method one was linear in the concentration range of 1 – 18 µg/mL
while linearity of the second method was found in the range of 1 – 14 µg/mL. Slope,
intercept and correlation coefficient of the first method were 0.0684, 0.0050 and 0.9998
respectively while for the second method, the same parameters were found to be 0.0864,
0.0027 and 0.9999 respectively. Molar absorptivity of the analyte in method one was
found to be 2.62 x 104 L/mol/cm while it was 3.25 x 104 L/mol/cm for the second
method. Sandell’s sensitivity for both the investigated methods was 0.01µg/cm 2/0.001A.
United States Pharmacopeia and International Conference on Harmonization’s guidelines
were followed in the application and validation of both methods. Limit of quantification
were found to be 0.312 µg/mL and 0.3 µg/mL for method one and method two
respectively. All the marketed products containing gatifloxacin as a major ingredient
were successfully analyzed by the proposed methods. Coefficient of variation of the
methods was found less than 2 % which indicate precision, accuracy and reproducibility
of the methods.
Salgado et al. (94) investigated a simple, sensitive and accurate spectrophotometric

method for gatifloxacin in bulk and marketed dosages. The gatifloxacin tablet solution
absorbs maximum at 287 nm. The method is linear in the concentration range of 4.0 –
14.0 µg/mL. Common additive of the marketed formulation do not affect the absorption
spectra and thus did not affect the quantification process.
Marona et al. (95) described a volumetric method for quantification of gatifloxacin in

dosage tablets. The method was found economical, simple, fast and precise. The method
is based on volumetric analysis of the analyte in glacial acetic acid with 0.1 M perchloric
acid. Validation of the method in terms of accuracy, precision and recovery was carried
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out and very good results were obtained. The method was found free of the interfering
effect of the common additives of dosage tablets.
Wishwanathan et al. (96) reported liquid chromatography/electrospray tandem mass

spectrometry for determination of gatifloxacin in samples of human plasma.
Ciprofloxacin was used as internal standard in the method. Gatifloxacin and the internal
standard were extracted from samples by solid phase extraction technique using Oasis ®
HLB. Triple quadrupole mass spectrometer was used for monitoring the precursor and
the degradation product of gatifloxacin. The spectrometer was coupled with positive ion
electrospray ionization in the multiple reaction monitoring modes. Mechanisms were
proposed for formation of dissociation products of gatifloxacin. Calibration curve was
linear in the concentration range of 10 – 1000 ng/mL with good correlation coefficient.
Precision of the method in terms of intra and interday was evaluated with the help of
coefficient of variation and found not more than 6 %. Accuracy of the method in terms of
percent error was less than 5.4 %. Detection limit of the method was found to be 500
pg/mL.
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References
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3. N. Sultana, M. S. Arayne, M. Akhtar, S. Shamim, S. Gul, M. M. Khan, "High-

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EXPERIMENTAL
RESULTS & DISCUSSION

Experimental
3.1 INVESTIGATION OF SPECTROPHOTOMETRIC METHOD FOR

DETERMINATION OF SPARFLOXACIN IN PHARMACEUTICAL
PREPARATIONS AND URINE SAMPLES
3.1.1 Method development strategy for the spectrophotometric determination of

sparfloxacin
A number of chemical substances change their optical characteristics with change in their
oxidation state. Sparfloxacin is yellow crystalline powder and its solution in
methanol/water is greenish pale yellow in colour. On oxidation it changes to reddish
colour product. It can be oxidized by a number of oxidizing agents. Ammonium
metavanadate in the presence of sulphuric acid acts as an oxidizing agent and can be used
for oxidation of sparfloxacin. Sparfloxacin changes its colour from greenish pale yellow
to reddish. This change in colour can be monitored and can be exploited for
determination of sparfloxacin. The proposed reaction mechanism of oxidation of
sparfloxacin by ammonium metavanadate is shown in figure 3.1.1.
Net Reaction Mechanism
88
Experimental
89
Experimental
Figure 3.1.1 The proposed reaction mechanism for oxidation of sparfloxacin by

vanadate ion
90
Experimental
3.1.2 Preliminary investigation of the possibility of oxidation of sparfloxacin by

ammonium metavanadate for its spectrophotometric determination
Preliminary studies were conducted to investigate the possible oxidation of sparfloxacin

by ammonium metavanadate in the presence of sulphuric acid. Initially high
concentration of sparfloxacin (100 µg/mL), ammonium metavanadate (0.1 M) and
relatively large volume of concentrated sulphuric acid were mixed in a beaker and heated
on boiling water bath for a minute or so to ensure oxidation of the drug. This reaction
resulted in a red colour product, indicating the successful oxidation of the drug by
ammonium metavanadate in the presence of sulphuric acid and subsequent
spectrophotometric determination of sparfloxacin. Further studies were focused on
optimization of various parameters leading to the complete oxidation of the drug.
3.1.3 Optimization studies for spectrophotometric determination of sparfloxacin

using ammonium metavanadate
After the preliminary reaction, it was evident that sparfloxacin could undergo oxidation
reaction with ammonium metavanadate to produce a red coloured product. The coloured
product could be determined by spectrophotometric method. For quantitative
determination of sparfloxacin, it was important to optimize various experimental
parameters of the method.
Instruments
Spectrophotometer (SP-300. Optima Inc.Tokyo Japan. Path length 1 cm), Digital

Analytical balance (Sartorius handy H51 Germany) and electric thermostatic water bath
(MC 02810175 Yu Jia China) were used during this investigation.
Reagents
All reagents used were of high grade purity, ammonium metavanadate (reagent grade
ACS, Scharlau), sulphuric Acid (95-98 % Merck), methanol (Merck). Standard reference
sparfloxacin was kindly gifted by Libra Pharmaceutical Industry pvt. Ltd. Peshawar,
Pakistan and commercial formulations of sparfloxacin (Sparaxin 100 mg, Abbot
91
Experimental
Laboratories Pakistan limited, Karachi Pakistan., Sparcin 100 mg, Fozan Pharmaceutical
Industries Pvt. Ltd. Peshawar Pakistan., Quspar 100 mg, The Schazoo Laboratories Pvt.
Ltd. Lahore Pakistan) were purchased from local market and were used during this work.
Solution preparation
Ammonium metavanadate solution
Ammonium metavanadate solution (0.04 M), was prepared by dissolving 1.148 g of

ammonium metavanadate in hot distilled water and transferred to 250 mL volumetric
flask and made the volume up to mark with distilled water.
Sulphuric acid solution
Sulphuric acid solution (7.0 M) was prepared by diluting 95.28 mL of conc. sulphuric
acid (95-98 % ) with cold distilled water and made the volume up to mark in 250 mL
volumetric flask.
Standard sparfloxacin solution
Standard sparfloxacin stock solution (1000 µg/mL) was prepared by dissolving 0.1 g of
authentic standard sparfloxacin in sufficient distilled methanol with vigorous shaking in
100 mL volumetric flask. Working standard solution (200 µg/mL) was prepared from the
above stock solution by dilution with distilled water.
Sample solution
Five tablets of each sample (Sparaxin 100 mg, Sparcin 100 mg and Quspar 100 mg)
were weighed separately to get the average weight of one tablet and were ground
together. The sample of the powdered tablets, claimed to contain 0.1 g sparfloxacin were
transferred to 100 mL beaker and 50 mL distilled methanol was added to it and
vigorously shaken to dissolve almost all the contents. The solution was transferred to 100
mL volumetric flask and made the volume up to mark with distilled methanol to obtain
1000 µg/mL sample solution. The solution was filtered and diluted with distilled water.
This solution was used as working sample solution during the work.
92
Experimental
Procedure
Aliquots of standard sparfloxacin solution (5.0 mL of 200 µg/mL solution) were

transferred to three separate 100 mL beakers. Then 15 mL of ammonium metavanadate
(0.04 M) was added followed by the addition of 10 mL of sulphuric acid (7.0 M) solution
to each beaker. These beakers were heated over boiling water bath for 30 seconds. On
cooling, the contents of the beakers were transferred to separate 50 mL volumetric flasks
and diluted up to the mark with distilled water. For optimization of wavelength, the
absorbance of the resultant red coloured product was measured from 490 to 550 nm and
each wavelength was calibrated with a reagent blank prepared using the same procedure
but without addition of the drug. The results are shown in figure 3.1.2.
For optimization of concentration of ammonium metavanadate, aliquotes of 5 mL of

standard sparfloxacin (200 µg/mL) solution were taken in nine separate 100 mL beakers.
To each beaker 10 mL of ammonium metavanadate in the concentration range of 0.01 –
0.05 M was added followed by the addition of 10 mL of sulphuric acid (7.0 M) and
heated over boiling water bath for 30 seconds. The contents were cooled and transferred
to separate 50 mL volumetric flasks and diluted up to the mark with distilled water.
Absorbance was measured at optimum wavelength (530 nm), the results are shown in
figure 3.1.3.
For optimization of volume of ammonium metavanadate (0.04 M), only volume of the
reagent was varied in the range of 3 – 21 mL and rest of the procedure was kept the same.
Absorbance was measured at 530 nm and the results are shown in figure 3.1.4.
To check the effect of sulphuric acid concentration, only molarity of sulphuric acid was
vaired in the range of 1 - 10 using 10 mL volume in each case. The colour was developed
in the same way as mentioned before and absorbance was measured at 530 nm. The
results are shown in figure 3.1.5.
For optimization of volume of sulphuric acid, only volume of sulphuric acid with
optimized molarity (7.0 M) was varied in the range of 2 – 20 mL and the colour was
93
Experimental
developed by the same procedure as mentioned before. Absorbance was measured at 530
nm. The results are shown in figure 3.1.6.
For optimization of heating temperture, optimized reagents were used in this procedure as
mentioned before, followed by heatin over electric thermostatic water bath in the
temperature range of 40 – 100 oC for 30 seconds. The contents were cooled and
transferred to 50 mL volumetric flask and made the volume up to mark with distilled
water. Absorbance was measured at 530 nm and the results are shown in figure 3.1.7.
Similarly heating time was optimized by heating the contents in the beakers over boiling
water bath and heating time was varied from 0.5 minute to 5 minutes. The beakers were
covered with watch glasses. The contents were cooled and transferred to 50 mL
volumetric flask and made the volume up to mark with distilled water. Absorbance was
measured and the results are shown in figure 3.1.8.
0.295
0.290
0.285
0.280
Absorbance
0.275
0.270
0.265
0.260
480 490 500 510 520 530 540 550 560
Wavelength (nm)
Figure 3.1.2 Wavelength optimization for the spectrophotometric determination of

sparfloxacin
94
Experimental
0.18
0.17
0.16
0.15
Absorbance 0.14
0.13
0.12
0.11
0.10
0 0.01 0.02 0.03 0.04 0.05 0.06
Conc. (M)
Figure 3.1.3 Effect of concentration of ammonium metavanadate on the

0.20
0.19
0.18
0.17
Figure 3.1.4 Effect of volume of ammonium metavanadate on the spectrophotometric

Absorbance
determination
0.16 of sparfloxacin
0.15
95
0.14
Experimental
0.20
0.19
0.18
Absorbance
0.17
0.16
0.15
0.14
0 2 4 6 8 10 12
Conc. (M)
Figure 3.1.5 Effect of concentration of sulphuric acid on the spectrophotometric

0.195
0.190
0.185
0.180
Figure 3.1.6 Effect of volume of sulphuric acid on the spectrophotometric
Absorbance
0.175
96
0.170
Experimental
0.190
0.185
0.180
Absorbance
0.175
0.170
0.165
30 40 50 60 70 80 90 100 110
Temperature (oC)
Figure 3.1.7 Effecting of heating temperature on the spectrophotometric determination

of sparfloxacin.
0.200
0.190
0.180
0.170
Figure 3.1.8 Effect of heating time on spectrophotometric determination of
Absorbance
sparfloxacin
3.1.4 Verification of 0.160

Beer’s law for determination of sparfloxacin by the proposed
spectrophotometric method
Instrument, reagents and solutions used were the same as before.

0.150
Procedure
0.140 97
Experimental
From standard stock solution of sparfloxacin (100 µg/mL), different volumes for 0.8 –
28.0 µg/mL were taken in ten separate beakers and optimum volume (15 mL) of
ammonium metavanadate (0.04 M) and 10 mL of sulphuric acid (7.0 M) were added and
heated for 30 seconds on boiling water bath. The beakers were covered with watch
glasses. The contents were cooled and transferred to 50 mL volumetric flask and made
the volume up to mark with distilled water. Absorbance of the coloured product was
measured at 530 nm against the reagent blank prepared by the same method without
addition of the drug. The results are given in table 3.1.1. A calibration plot of absorbance
against concentration of the drug was constructed and is shown in figure 3.1.9.
Table 3.1.1 Investigation of the effect of concentration on the absorption behavior of

sparfloxacin
Concentration (µg/mL) Absorbance

0.8 0.007
1.0 0.009
2.0 0.019
4.0 0.037
8.0 0.074
12.0 0.111
16.0 0.148
20.0 0.185
24.0 0.216
28.0 0.248
0.30
0.25
0.20
Absorbance
0.15
0.10
0.05
0.00
0 5 10 15 20 25 30
Conc. (µg/mL)
98
Experimental
Figure 3.1.9 Investigation of the effect of concentration on the absorption behavior of

sparfloxacin
Determination of detection and quantification limits for determination of

sparfloxacin by the proposed spectrophotometric method
Procedure
From standard stock solution of sparfloxacin (100 µg/mL), aliquots of 0.3 mL were taken
in ten separate beakers and optimum volume of ammonium metavanadate (0.04 M) and
sulphuric acid (7.0 M) were added and heated for 30 seconds on boiling water bath. The
beakers were covered with watch glasses. The contents were cooled and transferred to 50
mL volumetric flask and made the volume up to the mark with distilled water.
Absorbance of the coloured product was measured at 530 nm using Optima SP- 300
spectrophotometer against the reagent blank prepared by the same method without the
addition of the drug. Limit of detection and limit of quantification were calculated using
ten replicates analytes having lowest quantifiable concentration by the following
equations.
Limit of detection (LOD) (For concentration) = 3 x S
Limit of quantification (LOQ) (For concentration) = 10 x S
Where S = Standard deviation of the method
Standard deviation, relative standard deviation and molar absorptivity (ε) were calculated
and the results are summarized in table 3.1.2.
99
Experimental
Table 3.1.2 Analytical parameters for the spectrophotometric determination of

sparfloxacin
Characteristics Value
λmax (nm) 530
Beer’s law range (µg/mL) 0.8 – 28.0
Molar absorptivity (L/mol/cm) 4 × 103
Limit of detection (µg/mL) 0.15
Limit of quantification (µg/mL) 0.5
Regression equation Y = 0.009x + 0.002
Slope (b) 0.009
Intercept (c) 0.002
Correlation coefficient (r) 0.9996
Standard deviation (µg/mL) 0.05
Relative standard deviation (%) 8.77
3.1.5 Application of the investigated method for the analysis of sparfloxacin in

various pharmaceutical brands
A. The effect of common excipients found in commercial formulations on

determination of sparfloxacin using the proposed method
Procedure
In order to evaluate selectivity of the developed method for the analyses of

pharmaceutical preparations containing sparfloxacin, interferences effect of common
excipients added to commercial formulations was studied. For this purpose a known
concentration (10 µg/mL) of standard sparfloxacin was analyzed by the procedure as
mentioned before. Then the same concentration of standard sparfloxacin was taken in
separate beakers and different concentrations of the excipients were added in the ratio of
1:2.5, 1:5, 1:50 and 1:100. Optimized amount of ammonium metavanadate (15 mL of
0.04 M) and sulphuric acid was added to it and colour was developed in the same
100
Experimental
procedure as mentioned before. Absorbance of the coloured product was measured at 530
nm against the reagent blank. The experiments were performed in triplicate and mean
absorbance was calculated. The results are given in table 3.1.3 and are shown in figure
3.1.10.
Table 3.1.3 The effect of common excipients found in commercial formulations on

Excipient
Percent recovery
added
A B C D
Glucose 99.95 100.13 99.89 99.91
Sucrose 99.85 100.07 99.94 100.1
Sorbitol 100.15 100.1 99.85 100.12
Lactose 99.87 100.20 99.95 100.1
Starch 100.03 100.10 101.18 102.20
Talc 99.90 100.18 101.18 103.59
Where A, B, C and D is the percent recovery of the drug from the mixture of the drug and
excipient in the ratio of 1:2.5, 1:5, 1:50 and 1:100 respectively.
Figure 3.1.10 The effect of common excipients found in commercial formulations on

101
Experimental
B. Determination of precision and accuracy of the investigated method
Instrument, reagents and standard and sample solutions used were the same as before.
Procedure
Precision and accuracy of the investigated method was determined by analysis of three
separate sample solutions of the commercial formulations (Sparcin tablets, Quspar tablets
and Sparaxin tablets) at three different concentration levels. Calibration curve was drawn
by the same procedure as mentioned before and known concentrations (5, 10 and 15
µg/mL) of these sample solution were then analyzed by the proposed method in triplicate.
The results are shown in table 3.1.4.
Table 3.1.4 Evaluation of accuracy and precision of the proposed method for
sparfloxacin determination
Sample Amount taken Amount found Accuracy ± SD

(µg/mL) (µg/mL)
Sparcin tablets 5.0 5.0 100 ± 0.2
100 mg/tab 10 9.8 98 ± 0.16
15 14.7 98 ± 0.14
Quspar tablets 5.0 5.1 102 ± 0.2
100 mg/tab 10 10 100 ± 0.16
15 15 100 ± 0.14
Sparxin tablets 5.0 5.0 100 ±0.2
100 mg/tab 10 9.9 99 ±0.16
15 15 100 ± 0.14
102
Experimental
C. Percent recovery of sparfloxacin from commercial formulations (Standard

addition method)
Instrument, reagents and standard and sample solutions used were the same as before.
Procedure
For determination of percent recovery of sparfloxacin from commercial formulation, 5,

10 and 15 µg/mL standard sparfloxacin solution was mixed with preanalyzed 10 µg/mL
commercial sample of sparfloxacin. The colour was developed following the same
procedure as mentioned before. Blank solution was prepared following the same
procedure without addition of sparfloxacin. The absorbance of the resulting solution was
measured at 530 nm. The total amount of sparfloxacin in each fortified sample was found
from calibration curve. The experiments were performed in triplicate and the mean
percent recovery was calculated using the following formula. The results are given in
table 3.1.5.
Table 3.1.5 Evaluation of percent recovery of sparfloxacin from commercial

Samples Sample conc. Added Total conc. Recovery (%) ±

(µg/mL) (µg/mL) found (µg/mL) RSD
Sparcin 5.0 14.9 100.0 ± 0.20
Tablet 9.9 10.0 19.75 98.5 ± 0.15
100 mg/tab 15.0 24.80 99.3 ± 0.25
Quspar 5.0 14.8 100.0 ± 0.15
Tablet 9.8 10.0 19.6 98.0 ± 0.18
100 mg/tab 15.0 24.5 98.0 ± 0.14
Sparaxin 5.0 14.8 98.0 ± 0.15
Tablet 9.9 10.0 19.7 98.0 ± 0.25
100 mg/tab 15.0 24.9 100.0 ± 0.25
103
Experimental
D. Determination of active ingredients in the commercial formulations
Instrument, reagents and standard solutions used were the same as before.
Procedure
Three different commercial formulations that are Sparaxin 100 mg, Sparcin 100 mg and
Quspar 100 mg, were purchased from local market. Five tablets of each brand were
weighed separately and average weight of one tablet was found which are 0.285 g, 0.246
g, and 0.306 g for Sparaxin, Sparcin and Quspar respectively. Five tablets of each brand
were ground separately. The sample of the powdered tablets (average weight per tablet),
claimed to contain 100 mg sparfloxacin were transferred to 100 mL beaker and 50 mL
distilled methanol was added and vigorously shaken to dissolve almost all the contents.
The solution was transferred to 100 mL volumetric flask and made the volume up to the
mark with distilled methanol to obtain 1000 µg/mL sample solution. The solution was
filtered and working sample solution (100 µg/mL) was prepared by dilution with distilled
water. Known volumes (5 mL of 100 µg/mL) of this sample solution were then analyzed
by the proposed method in triplicate. The results are given in table 3.1.6.
Table 3.1.6 Determination of active ingredients in the commercial formulations
Brand name Active ingredient( mg/tab) t test value

Labeled value Found value (2.306)
Sparcin 100 99.9 ± 0.05 0.012
Quspar 100 100.0 ± 0.05 0.0
Sparaxin 100 100.0 ± 0.05 0.0
3.1.6 Validation of the proposed method for biological samples (Artificial Urine)
Instrument, reagents and standard solutions used were the same as before.
Preparation of artificial urine
104
Experimental
To check the applicability of the proposed method in different matrices, recovery test was
performed on spiked samples of artificial urine. Sample of artificial urine was prepared
according to the following table 3.1.7.
Table 3.1.7 Composition of artificial urine medium
Component Quantity (g)

Lactic acid 0.1
Citric acid 0.4
Sodium bicarbonate 2.1
Urea 10
Uric acid 0.07
Creatinine 0.8
Calcium Chloride. 2 H2O 0.37
Sodium Chloride 5.2
Iron (II) sulphate. 7 H2O 0.0012
Magnesium sulphate. 7 H2O 0.49
Sodium sulphate. 10 H2O 3.2
Potassium dihydrogen phosphate 0.95
Di-potassium hydrogen phosphate 1.2
Ammonium chloride 1.3
Distilled water To 1 Liter
Procedure
Aliquots of the methanolic solution of sparfloxacin containing 1, 5, and 10 μg/mL were

added to 10 mL of an artificial urine sample and shaken well. The same procedure was
followed for colour development as mentioned before and the content of sparfloxacin was
determined using standard calibration plot. Each result is average of the three replicates.
The results are shown in table 3.1.8.
Table 3.1.8 Evaluation of percent recovery of sparfloxacin from artificial urine

medium by the proposed method
Spiked amount Amount found Recovery (%)

(µg/mL) (µg/mL)
1.0 1.0 100.0 ± 0.33
5.0 4.9 98.00 ± 0.34
10.0 9.8 98.00 ± 0.82
105
Experimental
3.1.7 Results and discussion
A simple, rapid and sensitive spectrophotometric method has been developed with
application for determination of sparfloxacin in bulk, pharmaceutical formulations and
artificial urine. The method is based on oxidation of sparfloxacin by ammonium
metavanadate into red coloured product in the presence of sulphuric acid. Sparfloxacin is
yellow crystalline powder and its solution in methanol/water is greenish pale yellow in
colour. On oxidation it changes to red colour product. It can be oxidized using a number
of oxidizing agents ranging from mild to strong. Ammonium metavanadate in the
presence of sulphuric acid acts as an oxidizing agent and was used for oxidation of
sparfloxacin. Sparfloxacin changes its colour from greenish pale yellow to red. This
property provides an excellent and sensitive method for determining sparfloxacin.
The absorption spectrum of the red coloured product has an absorption maximum at 530
nm against the reagent blank (figure 3.1.2). For complete oxidation of sparfloxacin,
concentration of ammonium metavanadate solution was optimized (figure 3.1.3) and it
was found that absorbance of the analyte increases with increase in concentration of
ammonium metavanadate solution up to 0.04 M solution and remains constant after
further increase in concentration of the reagent. Similarly the effect of volume of
ammonium metavanadate (0.04 M) was also investigated (figure 3.1.4) and it was found
that with increase in volume of the reagent, absorbance of the analyte increases up to 15
mL and remains constant after further addition of the reagent. Thus 15 mL of 0.04 M of
ammonium metavanadate solution was found as optimal volume and concentration
respectively and was used throughout the analyses.
Ammonium metavanadate acts as oxidizing agent in acidic media and sulphuric acid was
used in the proposed method. The effect of concentration of sulphuric acid on the redox
reaction was investigated (figure 3.1.5). It was found that absorption of the analyte
increases with increase in concentration of sulphuric acid up to 7.0 M and decreases with
further increase in strength of the acid. Similarly the effect of volume of the acid (7.0 M)
was also investigated (figure 3.1.6) and it was found that with increase in volume of the
acid, absorbance of the analyte increases up to 10 mL and decreases with further addition
106
Experimental
of the acid. Therefore, 10 mL of 7.0 M sulphuric acid was used for oxidation of
sparfloxacin with ammonium metavanadate.
Redox reactions are mostly slow at room temperature and becomes fast at elevated
temperature. The effect of temperature on the proposed procedure was also investigated
(figure 3.1.7). The reaction mixture containing the drug, ammonium metavanadate and
sulphuric acid was heated on electric thermostat water bath from 40 oC up to boiling
water and it was found that absorbance of the analyte continuously increases with
increase in temperature up to the boiling of water in water bath. This is the maximum
possible temperature for the experiment as distilled water is used as a solvent and this
temperature was taken as optimal and further analyses were carried out at boiling water
bath. Heating time was also optimized (figure 3.1.8) and it was found that the reaction
completes just in 30 seconds on boiling water bath and gives maximum absorbance.
Decomposition of the analyte may take place at prolonged heating time and has negative
effect on absorbance behavior of the analyte, therefore, 30 seconds on boiling water bath
was taken as optimal heating time and was used throughout the analyses.
Analytical characteristics
The effect of concentration of sparfloxacin on the absorbance behavior was also

investigated (Table 3.1.1 and figure 3.1.9) at optimal conditions and a linear behavior
was observed in the concentration range of 0.8 – 28.0 µg/mL. The linearity of calibration
curve was proved by the high value of correlation coefficient (r = 0.9996). At optimum
conditions, the effect of concentration at lower level of sparfloxacin on the absorbance
behavior was also investigated to calculate standard deviation, relative standard
deviation, limit of detection and limit of quantification for the investigated method. The
results are summarized in table 3.1.2. Standard deviation and relative standard deviation
of the method were found to be 0.05 µg/mL and 8.7 respectively. Limit of detection
(LOD) and quantification (LOQ) of sparfloxacin by the proposed method were found to
be 0.15 µg/mL and 0.5 µg/mL respectively. The molar absorptivity was calculated and
was found to be 4 × 103 L/mol/cm.
Application
107
Experimental
In order to evaluate selectivity of the developed method for analyses of the

excipients added to commercial formulations was investigated and the results are given
table 3.1.3 and are shown in figure 3.1.10. It was found that the proposed method is free
of the interferences effect of common excipient present in the commercial formulations
of sparfloxacin except starch and talc. The tolerable concentration of both the excipient is
below 1:50 but within the formulation range.
The validity of the method was confirmed by applying standard addition procedure to
different commercial formulations of sparfloxacin (Table 3.1.5). In case of Sparcin,
quantitative recovery between 98.5 ± 0.15 % to 100.0 ± 0.20 % was obtained while in
case of Quspar and Sparaxin, quantitative recovery between 98.0 ± 0.14 % to 100.0 ±
0.15 % and 98.9 ± 0.25 % to 100.0 ± 0.25 % was obtained respectively.
To check the applicability of the proposed method for determination of sparfloxacin, the
method was successfully applied for the analysis of sparfloxacin in various samples of
commercial formulations and accuracy and precision of the method was evaluated. The
results are given in table 3.1.4 and 3.1.6. For all the formulations examined, the results of
the method were in good agreement with the labeled contents.
To check the applicability of the proposed method in biological samples, recovery test
was performed on spiked samples of artificial urine. A fixed volume of artificial urine
was spiked with varying concentration of the drug. In all cases quantitative recoveries
between 98.0 ± 0.34 % to 100.0 ± 0.33 % were obtained (Table 3.1.8).
108
Experimental
3.2 QUANTIFICATION OF SPARFLOXACIN IN PHARMACEUTICAL

DOSAGES AND BIOLOGICAL SAMPLES THROUGH CONDENSATION
REACTION WITH p-DIMETHYLAMINOBENZALDEHYDE (DMAB)

sparfloxacin
Sparfloxacin is yellow crystalline powder and its solution in methanol/water is greenish

pale yellow in colour which absorbs in the UV region. The optical characteristics of a
sparfloxacin can be changed with derivatization. A number of derivatives of sparfloxacin
can be prepared having absorption in visible region where it can be easily determined
quantitatively. Formation of Schiff’s base (a coloured product) is possible between amino
group of sparfloxacin and aldehyde group of DMAB in acidic medium. The product
could be determined spectrophotometrically, from which the amount of sparfloxacin
could be determined. The proposed reaction mechanism is shown in figure 3.2.1.
109
Experimental
Figure 3.2.1 The proposed reaction mechanism of sparfloxacin with DMAB
110
Experimental
3.2.2 Preliminary investigation of the possibility of condensation reaction between

sparfloxacin and DMAB for its spectrophotometric determination
Preliminary studies were conducted to investigate the possible formation of Schiff’s base
by the condensation reaction of sparfloxacin with DMAB in acidic medium. Initially high
concentration of sparfloxacin (100 µg/mL), DMAB (1.0 %) and relatively large volume
of concentrated sulphuric acid were mixed in a beaker and left at room temperature to
ensure complete reaction. This reaction resulted in a yellow colour product, indicating
successful condensation reaction of the drug with DMAB in the presence of sulphuric
acid and the subsequent spectrophotometric determination of sparfloxacin. Further
studies were focused on optimization of various parameters leading to the complete
reaction of the drug.
3.2.3 Optimization studies for spectrophotometric determination of sparfloxacin

using DMAB
Instruments
UV/Vis Spectrophotometer (Model SP-3000 plus, Optima Tokyo Japan), with matched 1
cm quartz cells for all spectrophotometric measurements, digital analytical balance
(Sartorius handy H51 Germany) and centrifugation of plasma and urine samples was
carried out on a clinical centrifuge (Model 800, China).
Reagents
All reagents used were of high grade purity. p-dimethylaminobenzaldehyde (Merck,

Germany), sulphuric acid (95-98 % Merck, Germany), and ethanol extra pure stabilized
(Riedel-deHaën, Seelze, Germany). Standard reference sparfloxacin was gifted by Libra
Pharmaceutical Industry pvt. Ltd. Peshawar Pakistan. Commercial formulations of
sparfloxacin (Sparaxin, Sparcin, Lowspar, and Quspar) were purchased from local
market.
111
Experimental
p-dimethylaminobenzaldehyde (0.3 %)
Solution of DMAB (0.3 %) was prepared by dissolving 0.15 g of DMAB in sufficient

ethanol and made the volume up to the mark in 50 mL volumetric flask.
Sulphuric acid solution (3.0 M)
Sulphuric acid solution (3.0 M) was prepared by diluting 39.5 mL of concentrated

sulphuric acid (95-98 %) with cold distilled water and made the volume up to mark in
250 mL volumetric flask.
Standard sparfloxacin solution (1000 µg/mL)
authentic standard sparfloxacin in 100 mL distilled ethanol with vigorous shaking in 100
mL volumetric flask. Working standard solution (200 µg/mL) was prepared from the
above stock solution by dilution with ethanol.
Sample solution (100 µg/mL)
Five tablets of each sample (Sparaxin 100 mg, Abbot Laboratories Pakistan limited,
Karachi Pakistan., Sparcin 100 mg, Fozan Pharmaceutical Industries Pvt. Ltd. Peshawar
Pakistan., Quspar 100 mg, The Schazoo Laboratories Pvt. Ltd. Lahore Pakistan,
Lowspar, 100 mg, Lowitt Lab New York USA.) were weighed to get the average weight
of one tablet and each brand was ground separately. After fine powdering of the tablets,
an accurate weighed portion of the sample equivalent to 0.01 g sparfloxacin was
dissolved in 30 mL extra pure ethanol and vigorously shaken. The solution was diluted
with same solvent in 100 mL flask and was filtered. The first 10 mL of the filtrate was
discarded.
Procedure
DMAB solution (1.0 mL of 0.3 %) was taken in 10 mL volumetric flasks followed by the
addition of H2SO4 (1.0 mL of 3.0 M) and 1.0 mL of the drug (100 µg/mL). The reaction
mixture was left for 20 minutes at room temperature for completion of the reaction. The
112
Experimental
mixture was diluted up to the mark in the flask with ethanol. Blank was prepared by the
same procedure without addition of the drug.
For optimization of wavelength, the absorbance of the resultant yellow coloured product
was scanned from 360 to 416 nm. The results are shown in figure 3.2.2.
For optimization of concentration of DMAB, aliquots of 1.0 mL of DMAB in the range

of 0.1 – 2.0 % were taken in 10 mL volumetric flasks and same procedure was followed
for obtaining the coloured product. Absorbance was measured at optimum wavelength
(392 nm) and the results are shown in figure 3.2.3.
To optimize volume of DMAB (0.3 %) solution, only volume of the reagent was varied
in the range of 0.25 – 3.0 mL and rest of the procedure was kept the same. Absorbance
was measured at 392 nm and the results are shown in figure 3.2.4.
To check the effect of concentration of sulphuric acid on the reaction, only molarity of
sulphuric acid soltion was vaired in the range of 0.0 – 5.0 M using 1.0 mL volume in
each case. The colour was developed in the same way as mentioned before and
absorbance was measured at 392 nm. The results are shown in figure 3.2.5.
mentioned before, followed by heating over electric thermostatic water bath. Heating
temperature was varied in the range of 25 – 100 oC for 20 minutes. Each experiment was
conducted in triplicate. The contents were cooled and transferred to 10 mL volumetric
flask and diluted up to the mark with ethanol. Absorbance was measured at 392 nm and
the results are shown in figure 3.2.6.
Similarly optimum reaction time and stability of the product was investigated by taking
the optimized reactants in flasks (10 mL) and were kept at room temperature and the
reaction time was varied from 10 – 60 minutes. The contents of the flasks were diluted up
to the mark with ethanol. Absorbance was measured against the reagent blank and the
113
Experimental
In order to study the effect of order of mixing the reactants on the yield of the reaction,
the optimized reagents were mixed in different order and the same procedure was
followed for colour developing as mentioned before. Absorbance was measured and the
results are given in table 3.2.1. The order of the reaction that gives highest absorbance
(DMAB + H2SO4 + Drug) was quite consistent with the theoretical bases of the proposed
reaction mechanism.
0.45
0.40
0.35
0.30
Absorbance
0.25
0.20
0.15
0.10
0.05
0.00
350 360 370 380 390 400 410 420
Wavelength (nm)

sparfloxacin
0.50
0.45
0.40
0.35
0.30
Figure 3.2.3 Effect of concentration of DMAB on the spectrophotometric
Absorbance
determination
114
0.20
Experimental
0.31
0.30
0.29
0.28
Figure 3.2.4 Effect of volume of DMAB (0.3 %) on the spectrophotometric
Absorbance
0.27
0.44
0.42
0.26
0.40
Absorbance
0.38
0.36
0.25
0.34
0 1 2 3 4 5 6
Conc. (M)
0.24
0.00 0.50 1.00 1.50 2.00
Volume (mL)
115
Experimental
0.34
0.32
0.30
Absorbance
0.28
0.26
0.24
0.22
0.20
20 30 40 50 60 70 80 90 100 110
Temperature (oC)
Figure 3.2.6 Effecting of heating temperature on the spectrophotometric determination

of sparfloxacin.
0.40
0.38
0.36
0.34
Figure 3.2.7 Effect of reaction time on spectrophotometric determination of

Absorbance
sparfloxacin
0.32
Table 3.2.1 Effect of the order of mixing of the reagents on absorbance behavior of
sparfloxacin
0.30
Order of addition absorbance
0.28
116
0.26
Experimental
DMAB + H2SO4 + Drug 0.461

Drug + H2SO4 + DMAB 0.458
Drug + DMAB + H2SO4 0.046
3.2.4 Verification of Beer’s law for determination of sparfloxacin by the proposed

spectrophotometric method
Instrument, reagent and solution were the same as mentioned before.
Procedure
Aliquots of 1.0 mL of DMAB solution (0.3 %) were taken in six separate volumetric
flasks (10 mL). To each flask 1.0 mL of H 2SO4 (3.0 M) was added followed by the
addition of varied volume of the drug (100 µg/mL) in the range of 0.25 – 8.0 mL
(corresponding to concentration range of 2.5 – 80 µg/mL) and kept for 20 minutes at
room temperature. The mixture was diluted up to the mark in the flasks with ethanol.
Blank was prepared by the same method without addition of the drug. Absorbance of the
analytes against the blank was measured at 392 nm. The results are given in table 3.2.2. A
calibration plot of absorbance against concentration of the drug was constructed and is
shown in figure 3.2.8.
Table 3.2.2 Investigation of the effect of concentration on the absorption behavior of
sparfloxacin
Conc. of the drug Absorbance

(µg/mL)
2.5 0.032
5 0.061
10 0.141
20 0.320
40 0.660
80 1.305
117
Experimental
1.40
1.20
1.00
0.80
Figure 3.2.8 Investigation of the effect of concentration on the absorption behavior of
Absorbance
sparfloxacin
For determination of limit

0.60 of detection (LOD) and limit of quantification (LOQ), aliquots
of 1.0 mL of DMAB solution (0.3 %) were taken in ten separate volumetric flasks.
Optimum volume of sulphuric acid (1.0 mL of 3.0 M) was added to each flask followed
by addition of sparfloxacin
0.40 with minimum absorbance (2.0 µg/mL) and allowed to
equilibrate for 20 minutes at room temperature. Absorbance of the coloured solution was
measured at 392 nm against the reagent blank. Standard deviation, relative standard
deviation (%), molar absorptivity,
0.20 limit of detection and limit of quantification were
calculated and the results are summarized in table 3.2.3. Limit of detection and limit of
quantification were calculated for ten replicates of sparfloxacin solution (2.0 µg/mL)
using the following formulae.
0.00
0 10 20 30 40 50 60
Limit of detection (LOD) (For concentration) = 3 x S Conc. (µg/mL)
Molar absorptivity for same concentration was calculated using the following formula.
Molar absorptivity (ε) = A/C × L
118
Experimental
Where A = absorbance
C = concentration (mol/L)
L = path length (cm)

sparfloxacin
Parameter Value
λmax (nm) 392
Beer’s law range (µg/mL) 2.0 – 80.0
Molar absorptivity (L/mol/cm) 4.9 × 103
Limit of detection (µg/mL) 0.22
Limit of quantification (µg/mL) 0.75
Regression equation (y) Y = 0.0124X
Slope (b) 0.0124
Intercept (a) 0
Standard deviation (µg/mL) 0.075
3.2.5 Effect of common excipients found in commercial formulations on

Instrument, reagent and standard solutions were the same as mentioned before.
Procedure

concentration (10 µg/mL) of standard sparfloxacin was analyzed by the procedure as
mentioned before. Then the same concentration of standard sparfloxacin was taken in
separate volumetric flasks and different concentrations of the excipients were added to it
in the ratio of 1:5, 1:10 and 1:50. Optimized amount of DMAB (1.0 mL of 0.3 %) and
sulphuric acid was added to it and colour was developed in the same way as mentioned
119
Experimental
before. Absorbance of the coloured product was measured at 392 nm against the reagent
blank and percent recovery of the drug was calculated. The experiments were performed
in triplicate and mean absorbance was calculated. The results are given in table 3.2.4 and
are shown in figure 3.2.9.
Table 3.2.4 Effect of common excipients on % recovery of the analyte using the
proposed method
Excipient added Percent recovery

A B C
Glucose 99.98 99.93 99.92
Sucrose 100.02 100.01 99.97
Sorbitol 100.1 100.05 99.95
Lactose 99.87 100.02 99.95
Starch 100.03 99.94 99.94
Talc 99.95 100.08 99.92
Mg. Stearate 99.97 99.98 99.99
Where A, B and C is the percent recovery of the drug from the mixture of the drug and
excipient in the ratio of 1:5, 1:10 and 1:50 respectively.
Figure 3.2.9 Effect of common excipients found in commercial formulations on

120
Experimental

A. Determination of precision and accuracy of the investigated method
Procedure
Precision and accuracy of the investigated method was determined by analysis sample
solutions of four commercial formulations (Sparcin tablets, Quspar tablet, Sparaxin
tablets and Lowspar tablets) at three different concentration levels. Calibration curve was
constructed using same procedure as mentioned before and known concentrations (10, 20
and 30 µg/mL) of these sample solutions were then analyzed by the proposed method in
triplicate. The results are shown in table 3.2.5.
Sample Amount taken Amount found Accuracy

(µg/mL) (µg/mL) ± RSD
10.0 9.90 99.0 ± 1.0
Sparcin (100 mg/tab) 20.0 20.0 100 ± 0.50
30.0 29.9 99.7 ± 0.33
10.0 9.87 98.7 ± 1.02
Quspar (100 mg/tab) 20.0 19.87 99.4 ± 0.03
30.0 29.83 99.4 ± 0.20
10.0 10.0 100 ± 0.70
Sparaxin (100 mg/tab) 20.0 19.9 99.5 ± 0.35
30.0 29.9 99.7 ± 0.23
10.0 9.73 97.3 ± 0.62
Lowspar (100 mg/tab) 20.0 19.7 98.5 ± 0.51
30.0 29.7 99.0 ± 0.24
121
Experimental
B. Percent recovery of sparfloxacin from commercial formulations (Standard

addition method)
Procedure
For determination of percent recovery of sparfloxacin from commercial formulation, 10,
20 and 30 µg/mL standard sparfloxacin solution was mixed with preanalyzed 10 µg/mL
commercial formulation sample of sparfloxacin. Colour was developed by following the
same procedure as mentioned before. The absorbance of the resulting solution was
measured at 392 nm. The total amount of sparfloxacin in each fortified sample was found
from the calibration curve. The experiments were performed in triplicate and the mean
percent recovery was calculated using the following formula. The results are given in
table 3.2.6.
Table 3.2.6 Evaluation of percent recovery of sparfloxacin from commercial

Samples Sample Added found Recovery ±

(µg/mL) (µg/mL) (µg/mL) RSD
10.0 19.90 100.0 ±
0.80
Sparcin (100 mg/tab) 9.9 20.0 29.90 100.0 ±
0.40
30.0 39.90 100.0 ±
0.17
10.0 19.86 99.3 ±
0.51
Quspar (100 mg/tab) 10.0 20.0 29.85 99.5 ± 0.54
30.0 39.80 99.5 ± 0.18
10.0 19.94 99.7 ± 0.51
122
Experimental
Sparaxin (100 mg/tab) 10.0 20.0 29.85 99.5 ± 0.57

30.0 40.0 100.0 ±
0.25
10.0 19.80 100.0 ±
0.36
Lowspar (100 mg/tab) 9.8 20.0 29.50 99.0 ± 0.24
30.0 39.52 99.3 ± 0.35
C. Determination of active ingredients in the commercial formulations
Instrument, reagent and solutions were the same as mentioned before.
Collection of samples
Four different commercial formulations that is Sparaxin 100 mg, Abbot Laboratories
Pakistan limited, Karachi Pakistan., Sparcin 100 mg, Fozan Pharmaceutical Industries
Pvt. Ltd. Peshawar Pakistan., Quspar 100 mg, The Schazoo Laboratories Pvt. Ltd.
Lahore Pakistan and Lowspar, 100 mg, Lowitt Lab New York USA were purchased
from local market.
Procedure
Five tablets of each brand were weighed separately and average weight of one tablet was
found which are 0.285 g, 0.246 g, 0.306 g and 0.294 g for Sparaxin, Sparcin, Quspar and
Lowspar respectively. Five tablets of each brand were ground separately. The sample of
the powdered tablets (0.0285 g, 0.0246 g, 0.0306 g and 0.0294 g for Sparaxin, Sparcin,
Quspar and Lowspar respectively), claimed to contain 0.01g sparfloxacin were
transferred to 100 mL beaker and was dissolved in 10 mL ethanol with vigorous shaking
to dissolve almost all the contents. The solution was diluted with the same solvent in 100
mL flask and was filtered. The first 10 mL of the filtrate was discarded. Known volumes
(2.0 mL of 100 µg/mL) of this sample solution were then analyzed by the proposed
method. Triplicate readings were taken in each case. The results are given in table 3.2.7.

Brand name Active ingredient (mg/tab) t-test value F-test value
Labeled value Found value (2.776) (9.28)
123
Experimental
Sparcin 100 99.95 ± 0.05 1.73 0.015

Quspar 100 100 ± 0.05 1.73 0.021
Sparaxin 100 100 ± 0.05 0.91 0.083
Lowspar 100 99.50 ± 0.08 0.81 0.028
3.2.7 Validation of the proposed method in spiked biological samples
performed on spiked samples of urine and human plasma.
Collection and preparation of samples
i. Urine sample
Urine sample was taken from three healthy volunteers at normal condition. 25 mL of the
samples were spiked with standard sparfloxacin (100 µg/mL) separately and centrifuged
at 3000 rpm for 15 minutes. The clear supernatant was separated and used in further
analysis.
ii. Plasma sample
Ten milliliter of plasma sample was separated from blood of three healthy volunteers at
normal conditions and was spiked with standard sparfloxacin (100 µg/mL). The samples
were deproteinated by addition of 10 mL of acetonitrile and centrifuged at 3500 rpm for
30 minutes. The clear supernatant was separated and used in further analysis.
Procedure
For validation of the proposed method in case of urine, from DMAB solution (0.3 %), 1.0
mL aliquots were taken in three different 10 mL volumetric flasks and 1.0 mL of H 2SO4
solution (3.0 M) was also added to each flask. Spiked urine samples (2.0 mL) were added
to each flask and diluted up to the mark with ethanol after 20 minutes at room
temperature. Blanks were prepared by the same procedure containing same volume of
124
Experimental
urine and without addition of the drug. Absorbance of the analytes against the blank was
measured at 392 nm and the content of sparfloxacin was determined using standard
calibration plot. Each result is average of the three replicates. The results are shown in
table 3.2.8.
Table 3.2.8 Spectrophotometric determination of sparfloxacin in spiked urine by the

proposed method
Sample Spiked urine

Amount added Amount found % Recovery ±
(µg/mL) (µg/mL) RSD
Sample 1 20.0 20.0 100 ± 0.33
Sample 2 20.0 20.2 101 ± 0.23
Sample 3 20.0 20.0 100 ± 0.33
In case of plasma, aliquots of 2.0 mL of the clear supernatant of the spiked plasma was
used for colour developing by following the same procedure as mentioned before and
absorbance was measured against reagent blank prepared by the same procedure
containing same volume of deproteinated plasma and without addition of the drug. The
content of sparfloxacin was determined using standard calibration plot. Each result is
average of the three replicates and are shown in table 3.2.9.
Table 3.2.9 Spectrophotometric determination of sparfloxacin in spiked plasma by the

proposed method
Sample Spiked plasma

Amount added Amount found % Recovery ±
Sample 1 20.0 20.0 100 ± 0.23
Sample 2 20.0 20.2 101 ± 0.23
Sample 3 20.0 20.0 100 ± 0.23
3.2.8 Determination of sparfloxacin by reference method (HPLC method)
125
Experimental
Sparfloxacin is a 3rd generation fluoroquinolone and is not yet in any Pharmacopeia and
thus has no standard method for the determination. However literature methods are
available. An HPLC method was selected and the proposed method was compared.
Instrument
An Acme 9000 Series HPLC equipped with SP930 isocratic pump, UV730D detector
Young Lin (Korea) was used. All separations were carried out on HiQ SiL (C 18 column
HS 4.6 × 15 mm KYATECH, Japan). A ks300 KUM SUNG ultrasonic (Korea) sonicator
was used as degasser.
A buffer solution of KH2PO4 (0.2 %) was prepared by dissolving 0.2 g of the salt in
distilled water and diluted the solution up to the mark in 100 mL volumetric flask.
Solutions of standard sparfloxacin and commercial formulations were prepared by the
same method as mentioned before. Mixture of 0.2 % KH 2PO4 (pH 3.2), cyanomethane
and methanol in the ratio of 40:30:30 was used as mobile phase for HPLC.
Procedure
HPLC system was equilibrated with the mobile phase prior to analysis. After
equilibration solutions of standard sparfloxacin in the concentration range of 2.0 – 10.0
µg/mL were injected one by one and the peak height was noted for each concentration. A
calibration curve of peak height versus concentration was constructed and is shown in
figure 3.2.10. Sample solutions of three commercial formulation in three different
concentration (2.0, 2.5 and 3.0 µg/mL) were injected to the system one by one and
accuracy and precision of the method were determined with three replicates. The results
are given in table 3.2.10.
126
Experimental
70
60
50
40
Figure 3.2.10 Calibration curve for sparfloxacin using reversed phase HPLC
Peak height (mV)
30
Table 3.2.10 Determination of sparfloxacin in commercial formulation and validation
by reference method
20 Mass (mg) determined

Name of commercial
Proposed Literature
formulation
method method
mean ± % RSD 99.91 ± 0.22 101.85 ± 1.78
10
Tab. Sparcin (100 mg/tab) F (9.28) 0.015
t (2.776) 1.73
mean ± % RSD 100 ± 0.33 99.81 ± 0.32
Tab. Sparaxin (100 mg/tab) F (9.28) 0.083
0
1 2 t (2.776) 3 0.91
4 5 6 7 8
mean ± % RSD 99.5 ± 0.17 99.97 ± 1.04
Tab. Lowspar (100 mg/tab) F (9.28) 0.028 Conc. (µg/mL)
t (2.776) 0.81
A simple, fast and accurate spectrophotometric method for the determination of

sparfloxacin in bulk, pharmaceutical formulation and biological samples using p-
dimethylaminobenzaldehyde (DMAB) has been developed and validated. Sparfloxacin
reacts with p-dimethylaminobenzaldehyde (DMAB) in acidic media and results in a
127
Experimental
yellow coloured product. The colour of the product is different from the colour of the
ethanolic solution of sparfloxacin and colourless ethanolic solution of p-
dimethylaminobenzaldehyde. The yellow coloured product is due to the condensation
reaction of sparfloxacin and DMAB reagent. The condensation reaction requires the
presence of an acid for the protonation of the carbonyl oxygen of the DMAB and
therefore leaving carbonyl carbon positively charged. Amino group of the sparfloxacin
then donates a lone pair of electrons to the positively charged carbon and internal
rearrangement results in the formation of condensation product (figure 3.2.1). This
property provides an excellent way for determination of sparfloxacin. Quantification of
the drug can be carried out by monitoring absorbance of the yellow coloured Schiff’s
base product using spectrophotometer.
The absorption spectrum of the yellow coloured product has an absorption maximum at
392 nm against the reagent blank (figure 3.2.2).
To establish the most favorable conditions for the condensation reaction, all variables
affecting the reaction like reagent concentration, molarity of sulphuric acid, and reaction
temperature were carefully studied and optimized.
Concentration of DMAB solution affects the reaction markedly. Its effect was studied in
the range of 0.1 – 2.0 % solution concentration. It was found that with increase in
concentration of DMAB from 0.1 – 1.0 %, absorbance of the analyte against the reagent
blank remains almost constant and decreases with further increase in concentration of the
reagent. Therefore, 0.3 % DMAB concentration was selected as an optimum reagent
concentration (figure 3.2.3). The effect of volume of DMAB solution on the absorbance
behavior of condensation product was also investigated in the range 0.25 – 3.00 mL.
Maximum absorbance was observed with 1.0 mL of DMAB solution (0.3 %) and thus
this volume was used throughout the analysis (figure 3.2.4).
Effect of acidity on the reaction was studied in the range of 0.0 – 5.0 M sulphuric acid
solution. Maximum absorbance of the condensation product against reagent blank was
obtained with 1.0 mL of 3.0 M H 2SO4 (Fig 3.2.5). Above or below the optimum
concentration of the acid, decrease in absorbance was observed. It indicates that at lower
128
Experimental
concentration, reaction is incomplete and higher acid concentration catalyzes breakdown

of the product, therefore, 1.0 mL of H2SO4 (3.0 M) was used throughout the analysis.
The effects of temperature and reaction time on the condensation reaction were studied in
the range of room temperature to 100 ˚C and from 10 – 60 minutes, respectively.
Maximum absorbance of the analyte against reagent blank was found at room
temperature (Fig 3.2.6) with 20 minutes reaction time (Fig 3.2.7). It appears that high
temperature causes decomposition of the condensation product and leads to decrease in
absorbance while at room temperature, the reaction occurs with moderate rate and the
product is quite stable for at least one hour.
The order of mixing of the reagent affects the nature of the reaction product and has
remarkable effect on absorption behavior. The effect of order of mixing of the reagent on
proposed method was investigated and the results are given in table 3.2.1. It was found
that adding sulphuric acid to the reagent followed by the drug gives maximum
absorbance indicating maximum yield of the condensation product as is proposed in the
reaction mechanism while on shuffling the order, decrease in absorbance was observed.
The effect of concentration of sparfloxacin on the absorbance behavior was investigated

(Table 3.2.2 and figure 3.2.8) at optimal conditions and a linear behavior between
concentration and absorbance was observed in the concentration range of 2.0 – 80.0
µg/mL. The linearity of calibration curve was proved by the high value of correlation
coefficient (r2 = 0.999). The limit of detection of the method was determined by
establishing the minimum level at which sparfloxacin can be detected reliably (3S) using
ten replicate determinations and limit of quantification was also calculated by
establishing the lowest concentration of sparfloxacin that can be measured with
acceptable precision and accuracy (10S) with ten replicate determinations. Precision of
the method was evaluated from the values of standard deviation and relative standard
deviation. The small values of standard deviation and relative standard deviation show
low scattering of the points on the calibration curve. Standard deviation and relative
standard deviation of the method were found to be 0.075 and 3.8 respectively. Limit of
129
Experimental
detection (LOD) and quantification (LOQ) of sparfloxacin by the proposed method were
found to be 0.22 and 0.75 µg/mL respectively. The molar absorptivity was calculated and
was found to be 4.9 × 103 L/mol/cm. The results are summarized in table 3.2.3.
Application
To evaluate the selectivity of the proposed method for analysis of the pharmaceutical
preparations containing sparfloxacin, the interferences effect of various pharmaceutical
additives such as glucose, lactose, sucrose, sorbitol, magnesium stearate, talc and starch
on the efficiency of the presented method were investigated. Solutions containing
sparfloxacin and one of the excipients taken separately at concentrations level five, ten
and fifty times greater than that of the sparfloxacin were analyzed by the proposed
method. Results are given in table 3.2.4 and are shown in figure 3.2.9. Recoveries of
sparfloxacin in selectivity study were found to be in the range of 97.0 ± 0.35 – 102.0 ±
0.79 % with relative standard deviation of 0.4 – 0.9 %. The result indicated that there
were no significant interferences produced by these excipients substances on the
proposed method for determination of sparfloxacin.
The repeatability of the proposed method was determined with three different
concentrations (10.0 – 30.0 µg/mL) of sparfloxacin commercial formulations in triplicate.
The results are summarized in table 3.2.5. The relative standard deviations (RSD) were
between 0.03 % and 1.02 % which are less than 2 %. The data indicate good repeatability
of the proposed method.
The accuracy of the proposed method in dosage form of four different brands of tablets
was evaluated by standard addition method. For this, known quantities of sparfloxacin in
the range of 10.0 – 30.0 µg/mL were added to definite amounts (10.0 µg/mL) of pre-
analyzed sparfloxacin in commercial formulations and the mixture was analyzed by the
proposed method. The average percent recoveries obtained were in the range of 99.3 –
100.0 % with relative standard deviation in the range of 0.0 – 0.8 which is less than 1.0 %
and shows good accuracy of the proposed method (table 3.2.6)
130
Experimental
The proposed method has been successfully applied to the determination of sparfloxacin
in commercial formulation. The results obtained for pharmaceutical dosage forms were
compared statistically with respect to accuracy by student t-test at 95 % confidence level
and precision by the variance ratio F-test with those of the literature HPLC method.
There was also no significant difference in precision between the proposed and literature
methods. The results show similar precision and accuracy in the analysis of
pharmaceutical dosage forms (table 3.2.7 and 3.2.10)
Good sensitivity as shown by the LOQ value, high precision and accuracy, as revealed by
the repeatability and recovery study obtained by the proposed method enabled the
determination of sparfloxacin in urine and plasma samples. Sparfloxacin is orally
administered at doses of 100 mg two times daily, which results in a urine level of
concentration of about 2.0 – 4.0 µg/mL. A fixed volume of urine and plasma was spiked
with varying concentration of the drug. In all cases quantitative recoveries between 100.0
% ± 0.33 to 101.0 % ± 0.23 were obtained (Table 3.2.8 and 3.2.9). The results obtained
were precise and accurate.
131
Experimental
3.3 INDIRECT SPECTROPHOTOMETRIC METHOD FOR

DETERMINATION OF SPARFLOXACIN IN BULK AND DOSAGE
FORM USING Ce (IV)

sparfloxacin
Oxidation-reduction reactions have been used as the basis for the development of simple
and sensitive spectrophotometric methods for the determination of many pharmaceutical
compounds. Due to its high oxidation potential, Ce (IV) has been widely used as an
effective analytical reagent for the indirect determination of a number of drugs. Its
solution in acidic medium is yellow in colour and absorbs at 315 nm while its reduced
form Ce (III) is colourless. Sparfloxacin reacts with Ce (IV) in acidic medium, reduces
Ce (IV) to Ce (III). Quantification of the drug can be carried out by monitoring the
decrease in absorbance of Ce (IV) with the help of spectrophotometer. The present work
was carried out by exploiting this potential of Ce for the indirect determination of
sparfloxacin in bulk and pharmaceutical formulations. The possible redox reaction of the
drug with Ce (IV) is given below.
132
Experimental

ammonium cerium (IV) sulphate for its spectrophotometric determination

by Ce (IV) in the presence of sulphuric acid which led to the decolourization of the
reagent solution. Initially high concentration of sparfloxacin (10 µg/mL), Ce (IV) (500
µg/mL) and relatively large volume of sulphuric acid (2.0 M) were mixed in a beaker
and heated on boiling water bath for 30 minutes to ensure oxidation of sparfloxacin and
reduction of Ce (IV). The disappearing of yellow colour Ce (IV) solution in the presence
of sulphuric acid indicated the possible reduction of Ce (IV) to Ce (III) and the
subsequent indirect spectrophotometric determination of sparfloxacin. Further studies
were focused on optimization of various parameters leading to the complete oxidation of
the drug and are given below.
3.3.3 Investigation of suitable parameter for the determination of sparfloxacin
Instruments
UV/Vis Spectrophotometer (Model SP-3000 plus, Optima Tokyo Japan), with matched
1.0 cm quartz cells, digital analytical balance (Sartorius handy H51 Germany) and
electric thermostatic water bath (MC 02810175 Yu Jia China) were used during this
investigation.
Reagents
All chemicals used were of high grade purity. Ammonium cerium (IV) sulfate dihydrate
(reagent grade ACS, Scharlau Barcelona Spain), sulphuric acid (95-98 % Merck,
Darmstadt Germany) and methanol (Merck, Darmstadt Germany) were used during this
work. Standard reference sparfloxacin was gifted by Libra Pharmaceutical Industry pvt.
133
Experimental
Ltd., Peshawar, Pakistan. Dosage formulations of sparfloxacin (Sparaxin, Sparcin and

Quspar) were purchased from local market.
Ammonium cerium (IV) sulfate dehydrate (500 µg/mL)
Ammonium cerium (IV) sulfate dihydrate [(NH 4)4 Ce(SO4)4.2H2O] solution was prepared
by dissolving 0.227 g in 1.5 M H 2SO4 with constant shaking. The contents were
transferred to 100 mL volumetric flask and made the volume up to the mark with the
same solvent.
Sulphuric acid (1.5 M)
Sulphuric acid solution was prepared by diluting 20.42 mL of concentrated sulphuric acid
(95 – 98 %) up to the mark with cold distilled water in 250 mL volumetric flask.
Standard sparfloxacin solution (100 µg/mL)
Standard sparfloxacin stock solution was prepared by dissolving 10.0 mg of authentic

standard sparfloxacin in 10.0 mL of methanol with vigorous shaking and finally diluted
to 100 mL with distilled water. Working standard solutions (10, 5.0, and 1.0 µg/mL) were
prepared fresh each day by dilution of the above solution with distilled water.
Sample preparation (100 µg/mL)
Pakistan., Quspar 100 mg, The Schazoo Laboratories Pvt. Ltd. Lahore Pakistan.) were
weighed separately and average weight of one tablet was found which are 0.285 g, 0.246
g, and 0.306 g for Sparaxin, Sparcin and Quspar respectively. The tablet forms of each
brand were ground separately. Sample of the powdered tablets equivalent to 0.01 g of
sparfloxacin was transferred to 100 mL beaker and dissolved in 10 mL distilled methanol
with vigorous shaking. The solution was transferred to 100 mL volumetric flask and
diluted up to the mark with distilled water to obtain 100 µg/mL sample solution. The
134
Experimental
solution was filtered and diluted with distilled water. This solution was used as working
sample solution during the work.
Procedure
Working standard sparfloxacin solution (0.5 mL of 5.0 µg/mL) was transferred to 100
mL beaker followed by the addition of 5.0 mL of ammonium cerium (IV) sulfate
dihydrate (500 µg/mL). The contents of the beaker were heated on boiling water for 15
minutes and on cooling, the contents of the beaker were transferred to 25 mL volumetric
flask and diluted up to the mark with distilled water. For wavelength optimization, the
absorbance of the Ce (IV) without redox reaction as a blank was scanned in the
wavelength range of 255 – 350 nm against the analyte using UV/Vis spectrophotometer
(Model SP-3000 plus, Optima Tokyo Japan). The results are shown in figure 3.3.1.
For optimization of concentration of cerium (IV), 1.0 mL of standard sparfloxacin (5.0

µg/mL) solution were taken in five separate beakers (100 mL). To each beaker varied
concentration of cerium (IV) in the concentration range of 100 – 500 µg/mL was added
and proceeded as mentioned before. Absorbance was measured at optimum wavelength
(315 nm) and the results are shown in figure 3.3.2.
For optimization of volume of cerium (IV) (500 µg/mL), only volume of the reagent was
varied in the range of 1 – 5 mL and rest of the procedure was kept same. Absorbance was
measured at 315 nm and the results are shown in figure 3.3.3.
To study the effect of concentraion of sulphuric acid, aliquots of optimized volume (5

mL) of cerium (IV) (500 µg/mL) prepared in solution of sulphuric acid varied in the
concentration range of 0.0 – 2.5 M were taken in six separate 100 mL beakers. To each
beaker, 1.0 mL of the drug (5.0 µg/mL) was added and followed the same general
procedure as mentioned before. Absorbance was measured and the results are shown in
figure 3.3.4.
For optimization of heating temperture of the reaction, optimized reagents were used
following the same procedure as mentioned before only varying heating temperature in
the range of 40 – 100oC on thermostatic water bath for 30 minutes. The cooled contents
135
Experimental
of the beakers were transferred to separate 25 mL volumetric flasks and diluted up to the
mark with distilled water. Absorbance was measured at optimum wavelength and the
Similarly for optimization of heating time, only the heating time was varied from 10 – 60
minutes. Absorbance was measured and the results are shown in figure 3.3.6.
1.20
1.00

sparfloxacin 0.80
Absorption
0.60
136
Experimental
3.00
2.50
Absorption
2.00
1.50
1.00
0.50
0.00
50 100 150 200 250 300 350 400 450 500 550
Conc. (µg/mL))
Figure 3.3.2 Effect of concentration of cerium (IV) on the spectrophotometric

3.00
2.50
2.00
Figure 3.3.3 Effect of volume of cerium (IV) on the spectrophotometric

Absorption
determination
137
1.00
Experimental
3.10
2.90
2.70
2.50
Absorbance
2.30
2.10
1.90
1.70
1.50
0.00 0.50 1.00 1.50 2.00 2.50 3.00
Conc. (M)

3.00
2.50
2.00
Absorbance
1.50
1.00
0.50
0.00
40 50 60 70 80 90 100 110
Temperature oC
Figure 3.3.5 Effect of heating temperature on the spectrophotometric determination of

sparfloxacin
138
Experimental
3.00
2.50
2.00
Figure 3.3.6 Effect of heating time on spectrophotometric determination of

Absorption
sparfloxacin
1.50
3.3.4 Verification of Beer’s law and determination of limit of detection and limit of
quantification
1.00
Instrument, reagent and solutions used were the same as above.
Procedure
0.50
From standard stock solution of sparfloxacin (1.0 µg/mL), different volumes for 0.02 –
0.2 µg/mL were taken in six separate beakers followed by addition of 5.0 mL of acidic
ammonium cerium (IV) sulphate (500 µg/mL) and heated for 30 minutes on boiling water
bath. The beakers were
0.00covered with watch glasses. The contents were cooled and
0
transferred to 25 mL volumetric flask and 10 20 up to the mark
made the volume 30with distilled 40
water. A blank solution was prepared by the same method without additionTime
of the
(min)drug
and its absorbance was measured against the analyte at 315 nm. The results are given in
table 3.3.1. A calibration plot of absorbance against concentration of the drug was
constructed and is shown in figure 3.3.7.
Table 3.3.1 Effect of concentration on the absorption behavior of sparfloxacin
Concentration
(µg/mL) Absorbance
139
Experimental
0.02 0.367
0.04 0.632
0.08 1.295
0.12 1.768
0.16 2.337
0.20 2.863
3.50
3.00
2.50
2.00
Figure 3.3.7 Effect of concentration on the absorption behavior of sparfloxacin
Absorption
Standard deviation and relative standard deviation (%) were calculated using ten
replicates of analytes having
1.50 lowest quantifiable concentration. The limits of detection
and quantification were calculated using the following equations.

1.00
Where S = Standard deviation

0.50 of the method
Molar absorptivity was calculated by using the following formula.
Molar absorptivity (ε) =0.00

A/C × L
0.00 0.05 0.10 0.15
Where A = absorbance Conc. (µg/mL)
C = concentration (mol/L)
140
Experimental
L = path length (cm)
Analytical parameters for spectrophotometric determination of sparfloxacin and its

statistical values are given in table 3.3.2.

sparfloxacin
λmax (nm) 315
Beer’s law limits (µg/mL) 0.02 – 0.2
Molar absorptivity (L/mol/cm) 3.24 × 106
Limit of detection (ng/mL) 1.6
Limit of quantification (ng/mL) 5.2
Regression equation Y = 8.98X + 0.018
Slope (b) 8.98
Intercept (c) 0.018
Correlation coefficient (r2) 0.999
Standard deviation (µg/mL) 5.2 × 10-4
3.3.5 Effect of common excipients found in dosage formulations on determination

of sparfloxacin using the proposed method
Procedure
In order to evaluate selectivity of the proposed method for the analyses of pharmaceutical
preparations containing sparfloxacin, interferences effect of common excipients added to
dosage formulations was investigated. For this purpose a known concentration (0.04
µg/mL) of standard sparfloxacin was analyzed by the procedure as mentioned before.
141
Experimental
Then the same concentration of standard sparfloxacin was taken in separate beakers and
different concentrations of the excipients were added in the ratio of 1:4, 1:8 and 1:12.
Optimized amount of ammonium cerium sulphate (5.0 mL of 500 µg/mL) was added to it
and the same procedure as mentioned before was followed for the analyses. Absorbance
of the analyte was measured at 315 nm. The experiments were performed in triplicate and
mean absorbance was calculated. The results are given in table 3.3.3 and are shown in
figure 3.3.8.
Table 3.3.3 Effect of common excipients on determination of sparfloxacin
Excipient added Percent recovery

A B C
Glucose 99.98 100.05 99.89
Sucrose 99.95 100.02 100.09
Sorbitol 100.01 100.03 100.08
Lactose 99.97 100.10 99.99
Starch 100.03 100.10 99.88
Talc 99.96 100.01 100.03
Mg. Stearate 100.07 99.87 99.97
Where A, B and C is the percent recovery of the drug from mixture of the drug and
Figure 3.3.8 Effect of common excipients on determination of sparfloxacin
142
Experimental

Procedure
Three different dosage formulations were purchased from local market. The samples of
the powdered tablets (0.0285 g, 0.0246 g and 0.0306 g for Sparaxin, Sparcin and Quspar
respectively), equivalent to 0.01g sparfloxacin were transferred to 100 mL beaker and 10
mL methanol was added and shaken vigorously to dissolve almost all the contents. The
contents were transferred to 100 mL volumetric flask and made the volume up to the
mark with methanol to obtain 100 µg/mL sample solution. The solution was filtered and
working sample solution (1.0 µg/mL) was prepared by dilution with distilled water.
Known volumes (2.0 mL of 1.0 µg/mL) of this sample solution were then analyzed by the
proposed method in triplicate. The results are given in table 3.3.4.
separate sample solutions of the dosage formulations (Sparcin tablets, Quspar tablets
and Sparaxin tablets) at three different concentration levels (0.04, 0.08 and 0.12 µg/mL).
Calibration curve was constructed by the same procedure as mentioned before and known
concentrations of these sample solution were then analyzed by the proposed method in
triplicate. The results are shown in table 3.3.5.
Table 3.3.4 Determination of active ingredients in the dosage formulations

Table Labeled value Found value (2.306) 3.3.5
Sparcin 100 98.67 ± 0.25 0.052
Quspar 100 99.67 ± 0.19 0.027
Sparaxin 100 100.0 ± 0.13 0
Evaluation of accuracy and precision of the proposed method for
143
Experimental
Sample Amount taken Amount found Accuracy ±

0.040 0.039 97.5 ± 1.82
Sparcin (100 mg/tab) 0.080 0.080 100.0 ± 0.89
0.120 0.119 99.17 ± 0.84
0.040 0.040 100.0 ± 1.77
Quspar (100 mg/tab) 0.080 0.080 100.0 ± 0.88
0.120 0.119 99.17 ± 0.60
0.040 0.040 100.0 ± 1.77
Sparaxin (100 mg/tab) 0.080 0.080 100.0 ± 0.88
0.120 0.121 100.83 ± 0.59
3.3.7 Percent recovery of sparfloxacin from dosage formulations (Standard

addition method)
Instrument, reagent and solutions used were the same as mentioned before.
Procedure
For determination of percent recovery of standard sparfloxacin from dosage formulation,

0.02, 0.04 and 0.08 µg/mL standard sparfloxacin solution was added to preanalyzed 0.04
µg/mL dosage sample of sparfloxacin. Oxidation of the drug was carried out using the
same procedure. The absorbance of the resulting solution was measured at 315 nm. The
total quantity of sparfloxacin in each fortified sample was found from calibration curve
constructed from standard sparfloxacin solution. Each experiment was performed in
triplicate and the mean percent recovery was calculated using the following formula. The
results are given in table 3.3.6.
Table 3.3.6 Evaluation of percent recovery of sparfloxacin from dosage formulations

by the proposed method
Samples Sample Added Total conc. Recovery
144
Experimental
(µg/mL) (µg/mL) found (µg/mL) (%)

0.02 0.059 95.0 ± 3.74
Sparcin (100 mg/tab) 0.04 0.04 0.081 102.5 ±
1.73
0.08 0.012 100 ± 0.88
0.02 0.06 100 ± 3.55
Quspar (100 mg/tab) 0.04 0.04 0.08 100 ± 1.78
0.08 0.119 98.75 ±
0.59
0.02 0.06 100 ± 3.55
Sparaxin (100 0.04 0.04 0.059 97.5 ± 1.82
mg/tab)
0.08 0.119 98.75 ±
1.26
A simple, rapid and sensitive spectrophotometric method has been developed with
application for determination of sparfloxacin in bulk and pharmaceutical formulations.
The method is based on oxidation of sparfloxacin by ammonium cerium (IV) sulphate.
Ce (IV), because of its high oxidation potential, has been widely used as an effective
analytical reagent for the indirect determination of a number of drugs. Its solution in
acidic medium is yellow in colour and absorbs at 315 nm while its reduced form, Ce (III)
is colourless. Sparfloxacin reacts with Ce (IV) in acidic medium, reduces Ce (IV) to Ce
(III). This property provides an excellent way for determination of sparfloxacin.
Quantification of the drug can be carried out by monitoring the decrease in absorbance of
Ce (IV).
The absorption spectrum of the ammonium cerium (IV) sulphate shows an absorption
maximum at 315 nm against sulphuric acid (1.5 M) (figure 3.3.1). Concentration of Ce
(IV) solution affects the reaction very markedly. According to the proposed mechanism,
Ce (IV) should be taken in excess to ensure the completion of the reaction with
sparfloxacin. By measuring the excess of Ce (IV) reagent, the consumed reagent would
correspond to the amount of sparfloxacin. The effect of Ce (IV) reagent concentration on
its reaction with sparfloxacin was investigated in the range of 100 – 500 µg/ mL (figure
145
Experimental
3.3.2). It was found that with increase in concentration of Ce (IV), absorbance of the
reagent blank against analyte continuously increases. Ce (IV) solution of 500 µg/mL was
selected for the proposed spectrophotometric method as it gives maximum absorbance
and has sufficient concentration for the reaction. The effect of volume of Ce (IV) solution
on the absorbance behavior of Ce (IV) was also investigated in the range 1 – 5 mL (figure
3.3.3) and 5 mL of Ce (IV) solution of 500 µg/mL was found to be optimum volume.
Therefore, 5 mL of Ce (IV) solution was used throughout the analysis.
Ammonium cerium (IV) sulphate acts as oxidizing agent in acidic media. The effect of
acidity on the reaction was studied in the range of 0.0 – 2.5 M sulphuric acid solution
(figure 3.3.4). Maximum absorbance of Ce (IV) was obtained with 1.5 M H 2SO4.
Decrease in absorbance was observed above and below the optimum concentration of the
acid.
The reaction between sparfloxacin and Ce (IV) was relatively slow at room temperature.
Therefore, the effect of temperature on the redox reaction was studied from 50 oC up to
boiling water temperature (100 oC). Maximum absorbance of the reagent blank against
the analyte was found at boiling water (figure 3.3.5). Boiling water temperature is the
maximum possible temperature for the reaction as distilled water was used as a solvent
and further analyses were carried out at boiling water bath. Heating time was also
optimized (figure 3.3.6) and it was found that the reaction completes in 30 minutes on
boiling water bath and gives maximum absorbance. Decomposition of the analyte is
expected at prolonged heating time and has negative effect on absorbance behavior of the
analyte; therefore, 30 minutes heating on boiling water bath was taken as optimum
heating time and was used throughout the analyses.
The effect of concentration of sparfloxacin on its absorbance behavior was investigated

concentration and absorbance was observed in the concentration range of 0.02 – 0.20
µg/mL. The linearity of calibration curve is evident from the high value of correlation
coefficient (r2 = 0.999). The limits of detection and quantification of the method were
146
Experimental
determined by establishing the minimum level at which sparfloxacin can be detected and
measured with acceptable precision with ten replicate determinations. Precision of the
method was evaluated from the values of standard deviation and relative standard
low scattering of the points on the calibration curve. The results are summarized in table
3.3.2.
Application
To evaluate the selectivity of the method for analysis of the pharmaceutical preparations
containing sparfloxacin, the interferences effect of various excipients were investigated.
Solutions containing sparfloxacin and one of the excipients taken separately in
concentrations four, eight and twelve times greater than that of the sparfloxacin were
analyzed by the proposed spectrophotometric method. A level of interference was
considered to be acceptable if the error was not higher than 3 % relative to the expected
sparfloxacin value. No interferences were observed in the determination of sparfloxacin
in the presence of the common excipients studied (Table 3.3.3 and figure 3.3.8). The
recovery of sparfloxacin was found to be 95 – 105 %.
In order to check the applicability of the proposed method for determination of

sparfloxacin, the method was successfully applied for analysis of sparfloxacin in various
samples of dosage formulations. The results are given in table 3.3.4. The precision of the
method was performed at three levels of concentration of each dosage formulation in
triplicate. The results are given in table 3.3.5. The percentage relative standard deviation
(% RSD) values were ≤ 1.82 showing good precision of the proposed method.
Accuracy of the proposed method was evaluated by standard addition method at three
different levels of concentration. The results are given in table 3.3.6. The recovery was
evaluated by comparing the agreement between the measured concentration of
sparfloxacin and known added concentration of sparfloxacin to the dosage formulations.
The recovery percentage value for Sparcin, Quspar and Sparaxin are between 95.0 and
102.5 with relative standard deviation in the range of 0.59 – 3.74 %. Closeness of the
results obtained to 100 % indicated good accuracy of the proposed method.
147
Experimental
For all the formulations examined, the results of the method were in good agreement with
the labeled contents. The validity of the method was confirmed by applying standard
addition method to different dosage formulations of sparfloxacin (Table 3.3.5). In all
cases quantitative recoveries between 95.0 ± 3.74 % to 102.0 ± 1.73 % were obtained.
3.4 INDIRECT SPECTROFLUORIMETRIC METHOD FOR

DETERMINATION OF SPARFLOXACIN IN BULK AND DOSAGE
FORM USING Ce (IV)
3.4.1 Method development strategy for the spectrofluorimetric determination of

sparfloxacin
Oxidation-reduction reactions have been used as the basis for the development of simple
and sensitive spectrofluorimetric methods for the determination of many pharmaceutical
compounds. Ce (IV), because of its high oxidation potential, has been widely used as an
effective analytical reagent for the indirect determination of a number of drugs. Its
solution in acidic medium is yellow in colour and is fluorescence inactive while its
reduced form Ce (III) is colourless and fluorescent. Sparfloxacin is fluorescence inactive
and could not be determined by direct spectrofluorometry. It could be reacted with Ce
148
Experimental
(IV) in acidic medium, reduces Ce (IV) to Ce (III). This reaction could be used for the
indirect fluorimetric determination of sparfloxacin, an analytical technique having the
basic advantage of considerably greater sensitivity as compared to spectrophotometry.
The present work was carried out by exploiting this potential of Ce for the indirect
determination of sparfloxacin in bulk and pharmaceutical formulations. The possible
redox reaction of the drug with Ce (IV) is given below.

ammonium cerium (IV) sulphate for its spectrofluorimetric determination

by Ce (IV) in the presence of sulphuric acid which led to the decolourization of the
reagent solution. Initially high concentration of sparfloxacin (10 µg/mL), Ce (IV) (500
µg/mL) and relatively large volume of sulphuric acid (2.0 M) were mixed in a beaker
and heated on boiling water bath for 30 minutes to ensure the reduction of the Ce (IV).
Yellow colour of the Ce (IV) solution disappeared in the presence of sulphuric acid
indicating the possible reduction of Ce (IV) to Ce (III) and the possibility of its
subsequent indirect spectrofluorimetric determination of sparfloxacin. Further studies
were focused on optimization of various parameters leading to the complete oxidation of
the drug and are given below.
3.4.3 Optimization studies for spectrofluorimetric determination of sparfloxacin

using Ce (IV)
After the successful preliminary reaction, it was evident that sparfloxacin could be
oxidized by Ce (IV) and get reduced to fluorescence active Ce (III). The fluorescence
intensity of the Ce (III) could be monitored using spectrofluorometer. For quantitative
determination of sparfloxacin, it was important to optimize various experimental
parameters of the method.
149
Experimental
Instruments
Spectrofluorophotometer, (RF 5301, Shimadzu, Japan) with short arc Xenon lamp (UXL
– 155) was used for the measurement of fluorescence intensity, digital analytical balance
(Sartorius handy H51 Germany) and electric thermostatic water bath (MC 02810175 Yu
Jia China) were used during this investigation.
Reagents
All chemicals used were of high grade purity. Ammonium cerium (IV) sulfate dihydrate
(reagent grade ACS, Scharlau Barcelona Spain), sulphuric acid (95-98 % Merck,
Darmstadt Germany) and methanol (Merck, Darmstadt Germany) were used during this
work. Standard reference sparfloxacin was gifted by Libra Pharmaceutical Industry pvt.
Ltd. Peshawar, Pakistan. Commercial formulations of sparfloxacin (Sparaxin, Sparcin
and Quspar) were purchased from local market.
Ammonium cerium (IV) sulfate dehydrate
Fresh ammonium cerium (IV) sulfate dihydrate [(NH 4)4 Ce(SO4)4.2H2O] solution (100
µg/mL) was prepared by dissolving 0.0454 g in 30 mL of H 2SO4 (1.0 M ) solution with
constant shaking. The contents were transferred to 100 mL volumetric flask and made the
volume up to the mark with the same solvent.
Sulphuric acid solution
Sulphuric acid solution (1.0 M) was prepared by diluting 13.62 mL of concentrated

sulphuric acid (95 – 98 %) up to the mark with cold distilled water in 250 mL volumetric
flask.
Standard sparfloxacin solution
150
Experimental
authentic standard sparfloxacin in 10 mL of distilled methanol with vigorous shaking and
finally diluted to 100 mL with distilled water. Working standard solutions (10, 5.0, and
1.0 µg/mL) were prepared by dilution of the above solution with distilled water.
Sample preparation
Pakistan., Quspar 100 mg, The Schazoo Laboratories Pvt. Ltd. Lahore Pakistan.) were
weighed separately to obtain average weight of one tablet which was found to be 0.285 g,
0.246 g, and 0.306 g for Sparaxin, Sparcin and Quspar respectively. The tablet forms of
each brand were ground separately. Sample of the powdered tablets equivalent to 0.01 g
of sparfloxacin was transferred to 100 mL beaker and dissolved in 10 mL distilled
methanol with vigorous shaking. The solution was transferred to 100 mL volumetric flask
and diluted up to the mark with distilled water to obtain 100 µg/mL sample solution. The
solution was filtered and diluted with distilled water. This solution was used as working
sample solution during the work.
Procedure
Working standard sparfloxacin solution (0.5 mL of 2.0 µg/mL) was transferred to 100
mL beaker followed by the addition of 4.0 mL of ammonium cerium (IV) sulfate
dihydrate (100 µg/mL). The contents of the beaker were heated on boiling water for 30
minutes. On cooling, contents of the beaker were transferred to 25 mL volumetric flask
and diluted up to the mark with distilled water. Wavelength for maximum excitation (λ ex)
and maximum emission (λem) for the fluorimetric behaviour of Ce (III) was investigated.
Excitation spectrum was obtained by scanning the analyte in the range of 215 – 295 nm
and emission spectrum was obtained by scanning the analyte in the range of 290 – 400
nm after excitation at 250 nm using spectrofluorophotometer and the results are shown in
figure 3.4.1.
151
Experimental
For optimization of concentration of ammonium cerium (IV) sulphate, aliquotes of 0.5

mL of standard sparfloxacin (2.0 µg/mL) solution were taken in five separate beakers
(100 mL). To each beaker 4.0 mL of ammonium cerium (IV) sulphate in the
concentration range of 50 – 300 µg/mL was added and the rest of the procedure was kept
the same as mentioned before. Fluorescence intensity was measured at optimum
wavelengths (λex 250 nm and λem 352 nm) and the results are shown in figure 3.4.2.
For optimization of volume of ammonium cerium (IV) sulphate (100 µg/mL), only
volume of the reagent was varied in the range of 1 – 5 mL and rest of the procedure was
kept same. Fluorescence intensity was measured at 352 nm and the results are shown in
figure 3.4.3.
To check the effect of concentraion of sulphuric acid solution, aliquots of optimized

volume of ammonium cerium (IV) sulphate (4.0 mL) prepared in varied concentration of
sulphuric acid in the range of 0.0 – 2.0 M were taken in six separate 100 mL beakers. To
each beaker, 0.5 mL of the drug (2.0 µg/mL) was added and followed by the same
general procedure as mentioned before. Fluorescence intensity was measured and the
To study the effect of heating temperture on the reaction, optimized amounts of the
reagents were taken in six separate beakers followed by heating over electric thermostatic
water bath in the temperature range of 50 – 100 oC for 30 minutes. The cooled contents of
the beakers were transferred to separate 25 mL volumetric flasks and diluted up to the
mark with distilled water. Fluorescence intensity was measured at optimum wavelengths
and the results are shown in figure 3.4.5.
Similarly for optimization of heating time, only the heating time was varied from 10 – 60
minutes, while keeping rest of the procedure the same. Fluorescence intensity was
measured and the results are shown in figure 3.4.6.
Stability of the final product Ce (III) and subsequent stability of the proposed method was
also investigated for two hour with 30 minutes interval. The results are shown in figure
3.4.7.
152
Experimental
Figure 3.4.1 Excitation (1) and emission (2) spectra of cerium (III) generated after
reaction with sparfloxacin
350
300
250
Fluorescence Intensity
153
200
Experimental
Figure 3.4.2 Effect of concentration of ammonium cerium (IV) sulphate on the

350
300
250
200
Figure 3.4.3 Effect of volume of ammonium cerium (IV) sulphate on the

150
100
50
0
0 1 2 3 4 5 6
Volume (mL)
154
Experimental
550
500
450
Figure 3.4.4 Effect of concentration of sulphuric acid on the spectrofluorimetric

400
250
200
150
100
350
50
0
40 50 60 70 80 90 100 110
Temperature (oC)
300
0.00 0.50 1.00 1.50
Conc. (M)
Figure 3.4.5 Effect of heating temperature on the spectrofluorimetric determination of
sparfloxacin
155
Experimental
500
450
400
Figure 3.4.6 Effect of heating time on spectrofluorimetric determination of sparfloxacin

350
440
420
400
380
360
300
340
320
300
0 30 60 90 120 150
250 Time (min)
Figure 3.4.7 Stability200

of fluorescence intenisty of Ce (III)
0 10 20 30 40
3.4.4 Effect of concentration of sparfloxacin on fluorescence intensity of Ce (III)
Time (min)
Instrument, reagent and solutions were the same as mentioned before.
Procedure
From standard working solution of sparfloxacin (1.0 µg/mL), different volumes for 0.02
– 0.1 µg/mL were taken in six separate beakers followed by addition of 4.0 mL of acidic
ammonium cerium (IV) sulphate (100 µg/mL) and heated for 30 minutes on boiling water
bath. The beakers were covered with watch glasses. The contents were cooled and
156
Experimental
transferred to 25 mL volumetric flask and made the volume up to the mark with distilled
water. Fluorescence intensity of the product was measured at 352 nm after excitation at
250 nm against a reagent blank. The blank was prepared by the same way without
addition of the drug. The results are given in table 3.4.1. A calibration plot of
fluorescence intensity versus concentration of the drug was constructed and is shown in
figure 3.4.8.
Table 3.4.1 Effect of concentration of the drug on the fluorometric behavior of Ce (III)
Concentration (µg/mL) FI
0.00 0.245
0.02 59.839
0.04 109.877
0.06 184.093
0.08 229.410
0.10 290.675
350
300
250
200
Figure 3.4.8 Effect of concentration of the drug on the fluorometric behavior of Ce (III)
replicates of analytes having
150 lowest quantifiable concentration. Limit of detection and
limit of quantification were calculated using the following equations.

100
157
50
Experimental
All the optical parameters and statistical values calculated for the proposed method are
given in table 3.4.2.
Table 3.4.2 Analytical parameters for the spectrofluorimetric determination of

sparfloxacin
λex(nm) 250
λem (nm) 352
Concentration range (µg/mL) 0.02 – 0.10
Limit of detection 3S (µg/mL) 0.006
Limit of quantification 10S
0.02
(µg/mL)
Regression equation (y) Y=2907.3X + 0.327
Slope (b) 2.91 × 103
Intercept (a) 0.327
Correlation coefficient (r2) 0.9975

A. Effect of common excipients found in commercial formulations on

Instrument, reagent and standard solution were the same as mentioned before.
Procedure

concentration (0.04 µg/mL) of standard sparfloxacin was analyzed by the same procedure
as mentioned before. Then standard sparfloxacin (0.04 µg/mL) was taken in separate
158
Experimental
beakers and different concentrations of the excipients were added maintaining drug:
excipient ratio of 1:2, 1:4 and 1:8. This was followed by addition of ammonium cerium
sulphate (4.0 mL of 100 µg/mL). Fluorescence intensity of the analyte was measured at
352 nm. The experiments were performed in triplicate and mean fluorescence intensity
was calculated. The results are given in table 3.4.3 and are shown in figure 3.4.9.
Table 3.4.3 Interferences effect of common excipients found in commercial

formulations on determination of sparfloxacin using the proposed method
Excipient
Percent recovery
Added
A B C
Glucose 99.98 99.94 100.01
Sucrose 99.99 99.98 99.95
Sorbitol 100.01 100.03 100.08
Lactose 99.97 99.95 99.99
Starch 100.02 99.97 100
Talc 100.01 100.01 99.98
Mg. Stearate 100.05 100.05 99.97
Where A, B and C is the percent recovery of the drug from the mixture of the drug and
Figure 3.4.9 Interferences effect of common excipients found in commercial

formulations on determination of sparfloxacin using the proposed method
159
Experimental
B. Precision and accuracy of the investigated method
Instrument, reagent, standard solution and sample solution were the same as mentioned
before.
Procedure
separate sample solutions of the commercial formulations (Sparcin tablets, Quspar
tablets and Sparaxin tablets) at three different concentration levels (0.02, 0.04 and 0.06
µg/mL). Calibration curve was constructed by the same procedure as mentioned before
and known concentrations of these sample solution were then analyzed by the proposed
method in triplicate. The results are shown in table 3.4.4.
Sample Amount taken Amount found Recovery ±

0.0200 0.0193 96.5 ± 6.00
Sparcin (100 mg/tab) 0.0400 0.0380 95.0 ± 1.86
0.0600 0.0593 98.8 ± 1.52
0.0200 0.0186 93.0 ± 3.12
Quspar (100 mg/tab) 0.0400 0.0383 95.7 ± 3.13
0.0600 0.0580 96.6 ± 1.72
0.0200 0.0182 91.0 ± 4.18
Sparaxin (100 mg/tab) 0.0400 0.0380 95.0 ± 1.32
0.0600 0.0580 96.6 ± 0.86
160
Experimental
C. Percent recovery of sparfloxacin from commercial formulations (Standard

addition method)
before.
Procedure
For determination of percent recovery of standard sparfloxacin from commercial

formulation, 0.02, 0.04 and 0.08 µg/mL standard sparfloxacin solution was added to
preanalyzed 0.02 µg/mL commercial sample of sparfloxacin and fluorescence intensity
was measured by following the same procedure as mentioned before. The experiments
were performed in triplicate and the mean percent recovery was calculated using the
following formula. The results are given in table 3.4.5.
Table 3.4.5 Evaluation of recovery (%) of sparfloxacin from commercial formulations

by the proposed method
Samples Sample Added Found Recovery

(µg/mL) (µg/mL) (µg/mL) (%) ± RSD
0.0200 0.0400 100.0 ±
2.54
Sparcin (100 mg/tab) 0.0200 0.0400 0.0561 93.5 ± 1.76
0.0600 0.0744 93.0 ± 2.66
0.0200 0.0404 101.0 ±
1.61
Quspar (100 mg/tab) 0.0200 0.0400 0.0557 92.8 ± 0.92
0.0600 0.0784 98.0 ± 0.26
0.0200 0.0404 101.0 ±
1.04
Sparaxin (100 mg/tab) 0.0200 0.0400 0.0607 102.2 ±
2.33
0.0600 0.0814 101.7 ±
161
Experimental
0.99
D. Determination of active ingredients in the commercial formulations
before.
Procedure
Three different commercial formulations were purchased from local market. The samples
of the powdered tablets (0.0285 g, 0.0246 g and 0.0306 g for Sparaxin, Sparcin and
Quspar respectively), equivalent to 0.01g sparfloxacin were transferred to 100 mL beaker
and 10 mL distilled methanol was added and shaken vigorously to dissolve almost all the
contents. The contents were transferred to 100 mL volumetric flask and made the volume
up to the mark with distilled methanol to obtain 100 µg/mL sample solution. The solution
was filtered and working sample solution (1.0 µg/mL) was prepared by dilution with
distilled water. Known volumes (1.0 mL of 1.0 µg/mL) of this sample solution were then
analyzed by the proposed method in triplicate. The results are given in table 3.4.6.

Sparcin 100 97.58 ± 0.25 0.15
Quspar 100 98.28 ± 0.19 0.27
Sparaxin 100 98.45 ± 0.13 0.11
A simple, rapid and sensitive spectrofluorimetric method has been developed with
application for determination of sparfloxacin in bulk and pharmaceutical formulations.
The method is based on reduction of cerium (IV) to fluorescent active Ce (III) by
sparfloxacin. This property of Cerium provides an excellent way for indirect
162
Experimental
determination of sparfloxacin. Quantification of the drug can be carried out by

monitoring the fluorescence intensity of the product Ce (III) with the help of
spectrophotofluorometer.
Optimum conditions were investigated for oxidation of the drug and the subsequent
monitoring of Ce (III). The excitation and emission spectrum of the product cerium (III)
shows an excitation wavelength at 250 nm and emission wavelength at 352 nm (figure
3.4.1).
The effect of Ce (IV) reagent concentration on its reaction with sparfloxacin was
investigated in the range of 50 – 300 µg/ mL (figure 3.4.2). Concentration of Ce (IV)
solution affects the reaction markedly and it was found that with increase in
concentration of Ce (IV), fluorescence intensity increases up to 150 µg/ mL of Ce (IV)
and remains almost constant with further increase in concentration of Ce (IV). The
fluorescence intensity of the produced Ce (III) is stable and reproducible when 100
µg/mL of Ce (IV) is used; therefore, this concentration was used for further analysis
although the signal is high at high concentration but was unstable. The effect of volume
of Ce (IV) solution on the fluorometric behavior of Ce (III) was also investigated in the
range 1 – 5 mL (figure 3.4.3) and 4 mL of Ce (IV) (100 µg/mL) solution was found to be
the optimum volume. Therefore, 4 mL of Ce (IV) solution was used throughout this
study.
Ammonium cerium (IV) sulphate acts as oxidizing agent in acidic media. The effect of
acidity on the reaction was studied in the range of 0.0 – 2.0 M of sulphuric acid solution
(figure 3.4.4). High fluorescence intensity was observed with small molar solution of
sulphuric acid but the results were neither stable nor reproducible, therefore, 1.0 M
H2SO4 was selected as optimum concentration for further analyses.
The reaction between sparfloxacin and Ce (IV) was relatively slow at room temperature.
Therefore, the effect of temperature on the redox reaction was studied in the range of
50 oC up to boiling water temperature (100 oC). Maximum fluorescence intensity of
Ce (III) was found at boiling water temperature (figure 3.4.5). Boiling water temperature
is the maximum possible temperature for the reaction as distilled water is used as a
163
Experimental
solvent and was taken as optimal for further analyses. Heating time was also optimized
(figure 3.4.6) and it was found that the reaction needs 30 minutes for completion on
boiling water temperature and gives maximum fluorescence intensity, therefore, 30
minutes on boiling water bath was taken as optimal heating time.
Stability of the redox reaction product Ce (III) was also investigated and it was found that
the reaction product is quite stable and gives constant signal up to two hours (figure
3.4.7).
The effect of concentration of sparfloxacin on the fluorescence behavior was investigated

concentration and fluorescence intensity was observed in the concentration range of 0.02
– 0.10 µg/mL. The linearity of calibration curve was proved by the high value of
correlation coefficient (r2 = 0.9975). The limit of detection of the method was determined
by establishing the minimum level at which sparfloxacin can be detected reliably (3S)
using ten replicate determinations and limit of quantification was also calculated by
establishing the lowest concentration of sparfloxacin that can be measured with
acceptable precision and accuracy (10S) with ten replicate determinations. Precision of
the method was evaluated from the values of standard deviation and relative standard
low scattering of the points on the calibration curve. The results are summarized in table
3.4.2.
Application
To evaluate the selectivity of the method for analysis of the pharmaceutical preparations
containing sparfloxacin, the interferences effect of various excipients were investigated.
Solutions containing sparfloxacin and one of the excipients taken separately in
concentrations two, four and eight times greater than that of the sparfloxacin were
analyzed by the proposed spectrofluorimetric method. A level of interference was
considered to be acceptable if the error was not higher than 3 % relative to the expected
164
Experimental
sparfloxacin value. No interferences were observed in the determination of sparfloxacin

in the presence of the common excipients studied (Table 3.4.3 and figure 3.4.9). The
recovery of sparfloxacin was found to be 94.9 ± 3.72 – 102.0 ± 1.94 %.
The validity of the method was confirmed by applying standard addition method to
different commercial formulations of sparfloxacin (Table 3.4.5) and the recovery was in
the range of 93.0 ± 2.66 % to 100. ± 2.54 %, 92.8 ± 0.92 % to 101.0 ± 1.61 % and 101.0
± 1.04 % to 102.2 ± 2.33 % for Sparcin, Quspar and Sparaxin respectively.
In order to check the applicability, accuracy and precision of the proposed method for
determination of sparfloxacin, the method was successfully applied for analysis of
sparfloxacin in various samples of commercial formulations. The results are given in
table 3.4.4 and 3.4.6. For all the formulations examined, the results of the method were in
good agreement with the labeled contents.
165
Experimental
3.5 MICELLAR-ENHANCED SPECTROFLUOROMETRIC

DETERMINATION OF MOXIFLOXACIN IN PHARMACEUTICAL
FORMULATIONS AND BIOLOGICAL SAMPLES
3.5.1 Method development strategy for the spectrofluorometric determination of

moxifloxacin
Quantitative spectrofluorometric methods are very useful primarily due to their enhanced
sensitivity and inherent selectivity. Fluorescence intensity can be enhanced by the
judicious use of micelles in the analytical scheme. This new approach provides increased
sensitivity, reduces the number of potential interferences and offers greater experimental
convenience. Moxifloxacin is native fluorescence in an aqueous solution and addition of
a micelle can increase the fluorescence intensity of the fluorophore. This fact could be
used to develop a sensitive method for the determination of moxifloxacin.
3.5.2 Preliminary investigation of the possibility of increase in fluorescence

intensity of moxifloxacin in the presence of sodium dodecyl sulphate (SDS)
Preliminary studies were conducted to investigate the possible increase in the

fluorescence intensity of moxifloxacin in the presence of different micelle such as SDS
(0.15 M), methyl cellulose (1.0 %), triton X-100 (1 %) and different buffer systems in the
pH range of 1.5 – 8.0. Aliquotes of 1.0 mL of the drug (1.0 µg/mL) and 5.0 mL of each
micellar with and without the addition of buffer solution were taken in 25 mL volumetric
flask and made the volume up to mark with distilled water. Fluorescence intensity of each
analyte was measured at the optimized wavelengths. The results are shown in figure 3.5.1
and 3.5.2.
166
Experimental
100
80
60
40
20
Figure 3.5.1 Fluorescence intensity of the analyte as a function of medium. Aq,

aqueous medium; PB, phosphate buffer (pH 3.5); CB, citrate buffer (pH
3.5); SDS, sodium dodecyl sulphate (0.15 M); MC, methyl cellulose (1.0
%); T - X100 = Triton - X100 (1.0 %)
45
40
35
Figure 3.5.2 Effect of pH on the fluorescence intensity of moxifloxacin
A hundred fold increase was observed in the fluorescence intensity of moxifloxacin in the
presence of SDS. The presence of micelle resulted in increased fluorescence intensity of
30
moxifloxacin indicating the subsequent spectrofluorometric determination of
moxifloxacin with high sensitivity. Further studies were focused on optimization of
various parameters leading to the development of a method for the determination of
moxifloxacin at lower concentration in biological samples.
25
167
Experimental
3.5.3 Optimization studies for spectrofluorometric determination of moxifloxacin

using micelle
After the preliminary reaction, it was evident that fluorescence intensity of moxifloxacin
could be increased by the use of micelle. For quantitative determination of moxifloxacin,
it was important to optimize various experimental parameters of the method.
Instruments
RF-5301 PC Spectrofluorophotometer, Shimadzu, Japan, equipped with 150 watt Xenon

discharge lamp, excitation, emission grating monochromators, and 1×1 cm quartz cell
was used for measurements of fluorescence intensity. The instrument was operated both
at low and high sensitivity with excitation and emission slit width set at 5 nm.
Centrifugation of plasma and urine sample was carried out on a clinical centrifuge
(Model 800, China with maximum speed 4000 rpm). Digital analytical balance (Sartorius
handy H51 Germany) was used for weighing the reagents and samples.
Reagents
All reagents used were of high analytical grade purity or if otherwise stated. Sodium
dodecyl sulphate (BDH Laboratory supplies, England) was used. Standard reference
moxifloxacin was provided by MKB Pharmaceutical Pvt. Limited Peshawar. Commercial
formulations of moxifloxacin (tablet Moxibact 400 mg/tab and infusion Moxibact 400
mg/250 mL, manufactured by S. J. and G. Fazul Ellahie (Pvt.) Ltd. Karachi Pakistan,
under Licence Continental Pharmaceuticals Karachi Pakistan, tablet Moxiget 400 mg/tab,
manufactured by Getz pharma (Pvt.) Ltd. Karachi Pakistan and Megamox 0.5 % eye
drops manufactured by Elko Organization (Pvt.) Ltd. Karachi Pakistan and marketed by
Sante (Pvt.) Limited Karachi Pakistan) were purchased from the local medicine store.
168
Experimental
SDS solution (0.15 M)
SDS (10.81 g) was dissolved in 100 mL distilled water and diluted up to 250 mL in
volumetric flask.
Standard moxifloxacin solution (50 µg/mL)
Standard moxifloxacin (0.0025 g) was dissolved in 10 mL distilled water with vigorous

shaking and made the volume up to the mark in 50 mL volumetric flask with distilled
water. 1.0 µg/mL working standard solution was prepared from the stock solution by
fresh dilution each day.
Sample solution
Five tablets of each commercial formulation were weighed separately and average weight
per tablet (0.595 g for Moxibact and 0.610 g for Moxiget) was calculated. They were well
crushed and grounded together using pestle and mortar. The sample of the drug powder
(0.0074 g of Moxibact and 0.0076 g of Moxiget) claimed to contain 0.005 g of
moxifloxacin were dissolved in sufficient distilled water and diluted with distilled water
in 50 mL volumetric flask to get 100 µg/mL stock solution of each sample. The solutions
were filtered and 1.0 µg/mL working sample solutions were prepared by dilution method.
For preparing stock solution of infusion and eye drops, 100 µg/mL sample solutions was
prepared by pipetting out sufficient volume directly and diluting with distilled water in 50
mL volumetric flask. Working sample solution (1.0 µg/mL) was prepared by further
dilution of the stock solution with distilled water.
Procedure
Aliquots of moxifloxacin (1.2 µg/mL) were taken in six separate 25 mL volumetric

flasks. SDS solution (5.0 mL of 0.15 M) was added to three of the flasks and the contents
of all six flasks were diluted with distilled water. Fluorometric behavior (λ ex and λem) of
the drug with and without the presence of micelle was investigated by scanning the
analyte solution for excitation spectra in the range of 271 – 320 nm and for emission
169
Experimental
spectra in the range of 471 – 520 nm using spectrophotofluorometer and the results are
shown in figure 3.5.3.
To check the effect of SDS concentration on fluorescence intensity of moxifloxacin,

aliquotes of 1.0 mL of the drug (1.0 µg/mL) and 5.0 mL of SDS from solutions having
molarity in the range of 0.0125 - 0.3 M were taken in 25 mL volumetric flask.
Fluorescence intensity of each analyte was measured at the optimized wavelengths. The
result is shown in figure 3.5.4.
For optimization of volume of SDS (0.15 %), only volume of the reagent was varied in
the range of 3.0 – 8.0 mL and rest of the procedure was kept the same. Fluorescence
intensity was measured at 494 nm and the results are shown in figure 3.5.5.
mentioned before, followed by heating on electric thermostatic water bath in the range of
room to bioiling water temperature for 10 minutes. The contents were cooled and
transferred to 25 mL volumetric flask and fluorescence intensity was measured at 494 nm
after excitaiton at 294 nm and the results are shown in figure 3.5.6.
100
80
60
40
20
0
250 300 350 400 450 500 550
Wavelength (nm)
drug only with SDS
Figure 3.5.3 Excitation and emission spectra of moxifloxacin with and without SDS
170
Experimental
120
100
80
Figure 3.5.4 Effect of concentration of SDS on the spectrofluorometric determination

of moxifloxacin
60
40
110
20
105
1000
0.00 0.05 0.10 0.15 0.20
Conc. (M)
Figure 3.5.5 Effect of volume of SDS (0.15 %) on the spectrofluorometric

determination
95 of moxifloxacin
90
171
Experimental
45
40
35
Figure 3.5.6 Effect of temperature on the fluorescence intensity of moxifloxacin

30
3.5.4 Effect of concentration of moxifloxacin on fluorescence intensity
Instrument, Reagent and Solutions used were the same as mentioned before.
Procedure 25
Aliquots of 5.0 mL of SDS solution (0.15 M) were added in a series of 25 mL volumetric

flasks and followed by the addition of varied concentration of moxifloxacin in the range
20
of 1.25 to 3200 ng/mL and diluted with distilled water. The fluorescence intensity of each
analyte in micellar system was measured at optimum wavelengths using PC RF-5301
spectrofluorophotometer against a reagent blank. All analyses were carried out in
triplicate and the results
15 were expressed as mean values. The results are given in table
20
3.5.1 and are shown in figure 3.5.7.30In order to40determine moxifloxacin
50 60 70
in biological 80
samples, the calibration plot of fluorescence intensity vs. concentration ofTemperature (oC)
moxifloxacin
was constructed for lower concentration of the drug and is shown in figure 3.5.8.
172
Experimental
Table 3.5.1 Effect of concentration on fluorimetric behaviour of moxifloxacin
Conc. ( µg/mL) Fluorescence intensity

0.00125 4.934
0.40000 28.563
1.20000 70.927
2.00000 120.997
2.80000 162.639
3.20000 177.734
200
150
100
50
0
0 0.5 1 1.5 2 2.5 3 3.5
Conc. (µg/mL
Figure 3.5.7 Effect of concentration on fluorimetric behaviour of moxifloxacin
1200
1000
800
600
400
200
0
0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4 0.45
Conc. (µg/mL)
Figure 3.5.8 Calibration curve for spectrofluorimetric determination of moxifloxacin

at lower concentration level
173
Experimental
Determination of limit of detection, limit of quantification and other analytical

characteristics of the proposed spectrofluorimetric method for determination of
moxifloxacin
Instrument, reagent and solutions used were the same as mentioned before
Procedure
Ten replicates with lowest quantifiable concentration of moxifloxacin (1.25 ng/mL)

which can be measured with acceptable precision and accuracy were analyzed by the
proposed method following the same optimized procedure as mentioned before. Limit of
detection (LOD), limit of quantification (LOQ) and other analytical characteristics were
calculated for the proposed method, and are given in table 3.5.2.
Table 3.5.2 Analytical characteristics for the spectrofluorometric determination of

moxifloxacin
Analytical parameter Value
λex (nm) 294

λem (nm) 494
Linear Range (ng/mL) 1.25 – 3200
Limit of detection (ng/mL) 0.186
Limit of quantification (ng/mL) 0.62
Regression equation (y) Y=54.462 X + 7.643
Slope (b) 54.462
Standard deviation (ng/mL) 6.2x10-2
174
Experimental
3.5.5 Investigation of interferences on determination of moxifloxacin using the

proposed method
Instrument, reagent and standard solution used were the same as mentioned before
Procedure
preparations containing moxifloxacin, the interferences effect of common excipients
added to commercial formulations was studied. For this purpose a known concentration
(0.04 µg/mL) of standard moxifloxacin was analyzed by the procedure as mentioned
before. Then the same concentration of standard moxifloxacin and different concentration
of the possible interferers in different concentration ratio of drug and excipient (1:50,
1:100 and 1:1000) were added to a series of volumetric flasks (25 mL) each containing
the optimized amount of SDS (5.0 mL of 0.15 M) and diluted up to the mark with
distilled water. The fluorescence intensity of each solution was measured at optimized
conditions. All experiments were performed in triplicates and the results are given in
Table 3.5.3 and are shown in figure 3.5.9.
175
Experimental
Table 3.5.3 Effect of common excipients found in commercial formulations on

determination of moxifloxacin (0.1 µg/mL) using the proposed method
Amount added
Excipient Ratio Recovery ± RSD
(µg)
5.00 50 100.17 ± 0.55
Glucose 10.0 100 99.28 ± 0.82
100 1000 100.96 ± 0.47
5.00 50 98.92 ± 0.12
Fructose 10.0 100 101.15 ± 0.42
100 1000 99.88 ± 0.64
5.00 50 98.16 ± 0.24
Sucrose 10.0 100 102.12 ± 0.35
100 1000 101.01 ± 0.55
5.00 50 100.43 ± 0.54
Sorbitol 10.0 100 102.01 ± 0.28
100 1000 101.41 ± 0.69
5.00 50 101.93 ± 0.46
Lactose 10.0 100 100.29 ± 0.68
100 1000 100.42 ± 0.65
5.00 50 100.59 ± 0.49
Starch 10.0 100 101.29 ± 0.56
100 1000 101.30 ± 0.78
5.00 50 99.42 ± 0.69
Talc 10.0 100 100.18 ± 0.86
100 1000 100.21 ± 0.38
5.00 50 99.83 ± 0.62
Mg. Stearate 10.0 100 100.81 ± 0.82
100 1000 100.76 ± 0.47
5.00 50 98.96 ± 0.45
Sod. Citrate 10.0 100 99.79 ± 0.34
100 1000 101.14 ± 0.68
5.00 50 100.28 ± 0.29
M. cellulose 10.0 100 99.85 ± 0.47
100 500 100.32 ± 0.66
176
Experimental
Figure 3.5.9 Effect of common excipients found in commercial formulations on

determination of moxifloxacin using the proposed method
Effect of some common cations typically found in human urine and blood samples on
fluorescence intensity of moxifloxacin was also investigated. For this purpose a known
concentration (0.1µg/mL) of standard moxifloxacin was analyzed. Then the same
concentration of standard moxifloxacin and different concentration of the possible cations
in different concentration ratio of drug and cations (1:50, 1:100 and 1:1000) were added
to a series of volumetric flasks (25 mL) followed by the addition of optimized amount of
SDS (5.0 mL of 0.15 M) and diluted up to the mark with distilled water. The fluorescence
intensity of each solution was measured and the results are given in table 3.5.4 and shown
in figure 3.5.10. Each experiment was performed in triplicates and mean percent recovery
was obtained.
177
Experimental
Table 3.5.4 Effect of common cations found in human plasma and urine samples on
Cation Amount added Ratio Recovery ± RSD

(µg)
Na+ 5.00 50 99.29 ± 0.95
10.0 100 101.42 ± 0.71
100 1000 99.95 ± 0.84
K+ 5.00 50 100.52 ± 0.54
10.0 100 101.35 ± 0.22
100 1000 99.54 ± 0.50
NH4+ 5.00 50 98.56 ± 0.99
10.0 100 100.92 ± 0.72
100 1000 102.11 ± 0.66
Ca++ 5.00 50 97.28 ± 0.50
10.0 100 100.59 ± 0.82
100 1000 99.23 ± 0.96
Mg++ 5.00 50 100.93 ± 0.64
10.0 100 99.29 ± 0.86
100 1000 101.22 ± 0.56
Zn++ 5.00 50 102.59 ± 0.94
10.0 100 101.29 ± 0.65
100 1000 100.57 ± 0.87
Cu++ 5.00 50 99.22 ± 0.96
10.0 100 100.81 ± 0.68
100 1000 99.29 ± 0.83
Fe++ 5.00 50 100.56 ± 0.54
10.0 100 101.39 ± 0.43
100 1000 100.19 ± 0.86
Al+++ 5.00 50 101.28 ± 0.92
10.0 100 102.45 ± 1.22
50.0 500 102.32 ± 1.50
Fe+++ 5.00 50 100.29 ± 1.11
10.0 100 101.52 ± 1.21
15.0 150 102.32 ± 1.35
178
Experimental
Figure 3.5.10 Effect of common cations found in human plasma and urine samples on
The effect of some common compounds typically found in human urine samples on the
fluorescence intensity of moxifloxacin was also investigated. For this purpose a known
concentration (0.1µg/mL) of standard moxifloxacin was analyzed and then the same
concentration of standard moxifloxacin and different concentration of the commonly
occurring compounds in human urine like urea, uric acid and citric acid in different
concentration ratio (1:50, 1:100 and 1:1000) were added to a series of volumetric flasks
(25 mL) followed by the addition of optimized amount of SDS (5.0 mL of 0.15 M) and
diluted up to the mark with distilled water. The fluorescence intensity of each solution
was measured and the results are shown in figure 3.5.11. Each experiments was
performed in triplicates and mean percent recovery was obtained. The results are given in
table 3.5.5.
179
Experimental
Table 3.5.5 Effect of common compounds occurring in human urine samples on

determination of moxifloxacin (0.1 µg/mL)
Compound Added (µg/mL) Ratio Recovery ± RSD

5 50 100.52 ± 0.939
Urea 10 100 101.25 ± 0.895
100 1000 99.93 ± 0.911
5 50 100.53 ± 0.749
Uric Acid 10 100 101.29 ± 0.631
100 1000 103.51 ± 0.937
5 50 100.47 ± 0.738
Citric acid 10 100 99.29 ± 0.699
100 1000 100.54 ± 0.517
Figure 3.5.11 Effect of common compounds occurring in human urine samples on

determination of moxifloxacin (0.1 µg/mL)
The effect of the presence of co-administered drugs such as Aspirin, Mefenamic acid,
Paracetamol, Vitamin C and Metronidazole on fluorescence intensity of moxifloxacin
was also investigated. For this purpose a known concentration (0.1µg/mL) of standard
moxifloxacin was analyzed by the procedure as mentioned before. Then the same
concentration of standard moxifloxacin and different concentration of the co-
administered drugs in different concentration ratio (1:5, 1:10 and 1:50) were added to a
180
Experimental
series of volumetric flasks (25 mL) followed by the addition of optimized amount of SDS
(5.0 mL of 0.15 M) and diluted up to the mark with distilled water. The fluorescence
intensity of each solution was measured at optimized conditions. Each experiment was
performed in triplicates and mean percent recovery was obtained. The results are given in
table 3.5.6 and are shown in figure 3.5.12.
Table 3.5.6 Effect of co-administered drugs on determination of moxifloxacin using

the proposed method (0.1 µg/mL)
Compound Added (µg/mL) Ratio Recovery ±

RSD
0.5 5 99.71 ± 0.86
Aspirin 1.0 10 98.53 ± 0.65
5.0 50 100.32 ± 0.79
0.5 5 101.43 ± 0.96
Mefenamic acid 1.0 10 100.05 ± 0.40
5.0 50 101.79 ± 0.14
0.5 5 98.99 ± 1.00
Paracetamol 1.0 10 99.01 ± 0.67
5.0 50 100.12 ± 0.48
0.5 5 100.09 ± 0.78
Vitamin C 1.0 10 99.55 ± 0.50
5.0 50 99.82 ± 0.40
0.5 5 100.69 ± 0.80
Metronidazole 1.0 10 100.11 ± 0.71
5.0 50 100.19 ± 0.10
181
Experimental
Figure 3.5.12 Effect of co-administered drugs on determination of moxifloxacin using

3.5.6 Application of the investigated method for the analysis of moxifloxacin in

A. Determination of precision and accuracy of the investigated method
Instrument, reagent, standard solution and sample solutions used were the same as
mentioned before.
Procedure
Precision and accuracy of the investigated method was determined by analysis of four
separate sample solutions of the commercial formulations (Moxibact tablets, Moxiget
tablet, Moxibact infusion and Megamox eye drops) at three different concentration levels.
Calibration curve was constructed using the same procedure as mentioned before and
known concentrations (0.04, 0.08 and 0.12 µg/mL) of these sample solution were then
analyzed by the proposed method in triplicate. The results are given in table 3.5.7.
182
Experimental
moxifloxacin determination (n = 3)
Sample Amount Amount found Recovery ±

taken (µg/mL) RSD
(µg/mL)
0.04 0.03977 99.42 ± 0.58
Tab. Moxibact 0.08 0.07970 99.62 ± 0.56
(400 mg/tab) 0.12 0.11797 98.31 ± 0.17
0.04 0.03942 98.55 ± 1.24

Tab. Moxiget 0.08 0.07966 99.58 ± 0.43
(400 mg/tab) 0.12 0.11940 99.50 ± 0.28
0.04 0.03947 99.34 ± 1.44

Inf. Moxibact 0.08 0.08003 100.04 ± 0.46
(400 mg/250 mL) 0.12 0.12001 100.01 ± 0.23
0.06 0.05966 99.43 ± 1.07
E. dps. Megamox 0.12 0.11989 99.91 ± 0.84
(500 mg/100 mL)
0.18 0.17999 99.99 ± 0.70
B. Percent recovery of moxifloxacin from commercial formulations (Standard

addition method)
mentioned before.
Procedure
For determination of percent recovery of moxifloxacin from commercial formulation,

0.04, 0.08 and 0.12 µg/mL standard moxifloxacin solution was mixed with preanalyzed
commercial sample of moxifloxacin (0.04 µg/mL). The fluorescence intensity of the
resulting solution was measured at 494 nm. The total amount of moxifloxacin in each
fortified sample was found from calibration curve. The experiments were performed in
triplicate and the mean percent recovery was calculated using the following formula. The
results are given in table 3.5.8.
183
Experimental
µg f ound in the f ortified sample - µg present in the sample

% Recovery = X 100
µg of standard added
Table 3.5.8 Evaluation of percent recovery of moxifloxacin from commercial

Samples Sample Added Found Recovery ±

(µg/mL (µg/mL) (µg/mL RSD
) )
Tab. Moxibact 0.04 0.0803 100.75 ± 0.74
(400 mg/tab) 0.04 0.08 0.1199 99.98 ± 0.59
0.12 0.1616 101.33 ± 0.53
Tab. Moxiget 0.04 0.0796 99.00 ± 1.41
(400 mg/tab) 0.04 0.08 0.1194 99.25 ± 0.63
0.12 0.1612 101.00 ± 0.42
Inf. Moxibact 0.04 0.0807 101.75 ± 0.71
(400 mg/250 0.04 0.08 0.1201 100.12 ± 0.65
mL)
0.12 0.1622 101.83 ± 0.87
E. dps. Megamox 0.06 0.0994 99.00 ± 0.57
(500 mg/100 mL) 0.04 0.12 0.1602 100.17 ± 0.73
0.18 0.2198 99.89 ± 0.34
mentioned before.
Procedure
Four different commercial formulations that is Moxibact tablets 400 mg, Moxiget tablets
400 mg, Moxibact infusion 400 mg/250 mL and Megamox eye drops, 500 mg/100 mL
were purchased from local market. Five tablets of each brand were weighed separately
and average weight of one tablet was found which are 0.595g and 0.610g for Moxibact
and Moxiget respectively. Five tablets of each brand were ground separately. The sample
of the powdered tablets (0.0074 g of Moxibact and 0.0076 g of Moxiget), claimed to
184
Experimental
contain 0.005 g moxifloxacin were transferred to 100 mL beaker and 20 mL distilled

water was added to it and vigorously shaken to dissolve almost all the contents. The
solution was diluted with distilled water in 50 mL flask and was filtered. The first 10 mL
of the filtrate was discarded and working sample solution (1.0 µg/mL) was prepared by
dilution method. For infusion and eye drops, 25 µg/mL sample solutions was prepared
by pipetting out sufficient volume directly and diluting with distilled water in 50 mL
volumetric flask. Working sample solution (1.0 µg/mL) was prepared by dilution method.
Known volumes (2.0 mL of 1.0 µg/mL) of this sample solution were then analyzed by the
proposed method in triplicate. The results are given in table 3.5.9.
Brand name Active ingredient

Labeled value Found value
Tab. Moxibact (mg/tab) 400 398.95 ± 0.58
Tab. Moxiget (mg/tab) 400 398.30 ± 0.43
Inf. Moxibact (mg/250mL) 400 400.03 ± 0.23
Megamox eye drops (mg/100 500 499.97 ± 0.70
mL)
D. Validation of the proposed method in spiked biological samples
a. Spiked urine sample
Instrument, reagent and standard solution used were the same as mentioned before.
Procedure
Urine samples from three healthy volunteers at normal conditions were taken and diluted
ten times with distilled water (5 mL of the urine were diluted to 50 mL with distilled
water). Moxifloxacin solution (1.0 µg/mL) was prepared with this solvent and
185
Experimental
centrifuged at 3000 rpm for 15 minutes. The supernatant was separated and 1 – 3 mL of
this solution was transferred to 25 mL volumetric flask, 5.0 mL of 0.15 M SDS solution
was added and made the volume up to the mark with distilled water to give final drug
concentration in the range of 0.04 – 0.12 µg/mL. Fluorescence Intensity of the solution
was measured at optimum conditions. The results are given in table 3.5.10.
Table 3.5.10 Spectrofluorometric determination of moxifloxacin in spiked urine by the

proposed method
Sample Spiked urine

Amount added Amount found Recovery ±
Urine 0.04 0.03973 99.32 ± 0.60
0.08 0.08022 100.28 ± 0.62
0.12 0.12004 100.03 ± 0.52
b. Spiked plasma sample
Procedure
Blood plasma was kindly provided by healthy volunteers and 10.0 mL of the plasma was
spiked with appropriate amount of the drug to give nominal concentration 1.0 µg/mL of
the drug. It was centrifuged at 3500 rpm for 20 minutes. The clear supernatant was taken.
No further treatment was carried out and this sample was directly used for further
analysis. Appropriate volumes (1 – 3 mL) of spiked plasma was transferred to 25 mL
volumetric flask and to this 5.0 mL of 0.15 M SDS solution was added and made the
volume up to the mark with distilled water to give final drug concentration in the range of
0.04- 0.12 µg/mL. Fluorescence intensity of the solution was measured and the results are
Table 3.5.11 Spectrofluorimetric determination of moxifloxacin in spiked plasma by the

proposed method

186
Experimental
0.04 0.04008 100.20 ± 0.50

Plasma 0.08 0.08005 100.06 ± 0.64
0.12 0.12022 100.18 ± 0.84
A simple, fast and accurate spectrofluorometric method for the determination of

moxifloxacin in bulk, pharmaceutical formulation and biological samples using sodium
dodecyl sulphate (SDS) as a micelle, has been developed and validated. Moxifloxacin is
native weak fluorescent and has been determined directly with linear range 30 to 300
ng/mL using 287 nm as excitation and 465 nm as emission wavelengths (Ocaña et al.,
2000). Its fluorescence intensity can be enhanced several times by the use of SDS with a
slight shift in the λex and λem (figure 3.5.1). This enhanced signal extended the linear range
at both side and made the measurement of even trace quantity of moxifloxacin possible.
This property provides an excellent way for determination of moxifloxacin.
Quantification of the drug with enhanced sensitivity could be carried out by monitoring
micellar enhanced fluorescence intensity of moxifloxacin.
To establish the most favorable conditions for the enhanced fluorescence intensity of
moxifloxacin, all variables affecting the reaction such as nature and concentration of
micellar media, pH of the medium and reaction temperature were carefully studied and
optimized.
The fluorimetric properties of moxifloxacin were investigated in the presence of different

micelle such as SDS (0.15 M), methyl cellulose (1.0 %), triton X-100 (1.0 %) and in
different buffers such as phosphate and citrate. It was observed that fluorescence intensity
of moxifloxacin enhanced by 100 fold in the presence of SDS (figures 3.5.4) as compared
to aqueous solution and other micellar media.
Concentration of SDS solution affects the reaction markedly. Its effect was studied in the
range of 0.0125 to 0.3 M and it was observed that with increase in concentration of SDS
up to 0.1 M, fluorescence intensity of the analyte increase. This enhancement in
fluorescence phenomenon may be due to protection of lower excited state of fluorescent
compound from quenching process in SDS micellar that can readily occurs in aqueous
187
Experimental
solutions. Above 0.1 M SDS concentration, the fluorescence intensity remained constant.
Therefore, SDS concentration 0.15 M was selected as optimum reagent concentration for
the analysis (figure 3.5.2). The effect of volume of 0.15 M SDS solution on the
fluorescence intensity of the analyte was also investigated in the range of 3 to 8 mL and 5
mL was found as the optimum volume for best results (figure 3.5.3).
The effect of pH and of different buffer systems on the fluorescence intensity of 0.04
μg/mL moxifloxacin solutions was studied and it was observed that at lower pH, the
fluorescence intensity is high (figure 3.5.5). As moxifloxacin solution is acidic itself due
to the presence of carboxyl group, therefore further analysis were performed without any
buffer solution.
Increase in temperature has negative effect on the fluorescence intensity of the analyte.
Generally, it happens with fluorescent compounds due to internal changes in structure of
molecules and external conversion process. Good results were obtained at room
temperature (figure 3.5.6) and thus making the process easier; further analyses were made
on room temperature.
Under the optimum experimental conditions of the proposed method, a linear response
between the fluorescence intensity and concentration of moxifloxacin was observed in
the concentration range of 1.25 to 3200 ng/mL. The linearity of calibration curve was
proved by the high value of correlation coefficient (r 2 = 0.9997). The linear regression
equations, slopes, intercepts, correlation coefficients, standard deviation and relative
standard deviation of the response factors are given in table 3.5.2. The limit of detection
(LOD) and limit of quantification (LOQ) were calculated using signal/noise (S/N) ratio
method. The LOD value was calculated where S/N was 3 and LOQ value where S/N was
10 and it was found to be 0.186 and 0.62 ng/mL, respectively.
Application
To evaluate the selectivity of the developed method for the analysis of pharmaceutical
preparations containing moxifloxacin, the interferences effect of various pharmaceutical
188
Experimental
additives such as glucose, fructose, lactose, sucrose, sorbitol, magnesium stearate, sodium
citrate, methyl cellulose, talc and starch was studied. Solutions containing moxifloxacin
and one of the excipients taken separately in concentrations fifty, hundred and thousand
times greater than that of the moxifloxacin were analyzed by the proposed method. A
level of interference was considered to be acceptable if the error was not higher than 3%
relative to the expected moxifloxacin value. The results indicated that there was no
significant interferences effect by the excipients studied on the fluorimetric behaviour of
moxifloxacin (table 3.5.3 and figure 3.5.9). Fluorescence intensity of moxifloxacin was
also measured in the presence of some common cations typically found in urine and
blood plasma. The micellar enhanced fluorescence method was found free from the
interferences effect of these ions except Al +++ and Fe+++ which decrease fluorescence
behaviour of moxifloxacin at concentration level greater than 50 µg/mL for Al +++ and
greater than 15 µg/mL for Fe+++ respectively (table 3.5.4 and figure 3.5.10). This decrease
in fluorescence intensity is mainly due to complex formation of the drug with these
cations at higher concentration. The major constituents of urine such as urea, uric acid
etc. also do not have any interference effect on the method. Fluorescence intensity of
moxifloxacin was measured in the presence of these compounds occurring in urine and
found good percent recovery (table 3.5.5 and figure 3.5.11). Mostly antibiotics are not
given alone, some antipyretic, analgesic or vitamins are given along with the antibiotics
to the patient and thus the effect of presence of these co-administered drugs on the
determination of moxifloxacin was studied. It was observed that the studied co-
administered drugs do not affect fluorescence behaviour and therefore, good percent
recovery was obtained (table 3.5.6 and figure 3.5.12).
The precision and accuracy of the proposed method was checked by determining
moxifloxacin concentration in replicate for each concentration within the linear range of
concentration. The results are summarized in table 3.5.7. The relative standard deviation
(RSD) considered being very satisfactory and the recovery test was found to be in the
range of 98.31 to 99.62 %, 98.55 to 99.58 %, 99.34 to 100.04 % and 99.43 to 99.99 % for
Moxibact tablets, Moxiget tablets, Moxibact infusion and Megamox eye drops
respectively which indicates a good accuracy.
189
Experimental
The validity of the proposed method in dosage form of four different brands was tested
for possible interference using standard addition method. The average percent recoveries
obtained were in the range of 99.00 ± 1.41 % to 101.83 ± 0.87 % (table 3.5.8) which
shows accuracy of the proposed method for determination of moxifloxacin in commercial
pharmaceutical preparations. The proposed method has been successfully applied for the
determination of moxifloxacin in commercial preparations (tablets, infusion and eye
drops). For all the formulations examined, the results obtained for pharmaceutical dosage
forms (table 3.5.9) were in good agreement with the label claimed value.
Good sensitivity obtained by the proposed method suggested the quantification of

moxifloxacin in urine and plasma samples. Moxifloxacin is orally administered at doses
of 400 mg once a day, which results in plasma level of concentration of about 0.3 to 3.0
μg/mL. For accurate determination of moxifloxacin in biological samples, standard
addition method was used for quantitative determination of moxifloxacin in urine and
plasma samples. In case of urine, percent recovery was found to be 99.88 ± 0.59 % while
in case of plasma, it was found 100.15 ± 0.08 %. The results obtained are satisfactorily
precise, accurate and are given in table 3.5.10 and 3.5.11. The method can be used for
determination of moxifloxacin concentration in biological samples in clinical analysis.
190
Experimental
3.6 SENSITIVE AND RAPID SPECTROFLUORIMETRIC METHOD FOR

DETERMINATION OF FLUOROQUINOLONES BY CHARGE-
TRANSFER COMPLEXATION WITH CHLORANILIC (CL) ACID
3.6.1 Method development strategy for the spectrofluorometric determination of

fluoroquinolones
Most of the fluoroquinolones are native fluorescent. Levofloxacin, moxifloxacin,

gatifloxacin and ciprofloxacin are important members of this group which show weak
fluorescence activity. Fluorescence signal of these pure fluoroquinolones in aqueous
medium is very weak and cannot be determined directly. However different solvents may
have positive effect and micellar medium has been used for enhancement of the
fluorescence intensity and such factors have been exploited for determination of
fluoroquinolones.
The molecular interaction between pair of electrons donors and pair of electrons
acceptors (Lewis acid–base reaction) are generally associated with the formation of
charge-transfer (CT) complexes. Charge-transfer complexes affect the absorption
behavior of the molecule and colourless substances become coloured and non-fluorescent
turn into fluorescent and results in enhancement of fluorescence activity. The formation
of CT complex can be rapidly assessed for its validity as a simple quantitative analytical
method for many drug substances, which can act as electron donors. Chloranilic acid is π
electron acceptor and readily forms charge-transfer complexes with basic nitrogenous
compounds as electrons donors. Fluoroquinolones has piperazine ring containing basic
nitrogen and is able to form a charge-transfer complex with π electrons acceptors like
chloranilic acid. This reaction could be exploited for fluorimetric determination of
fluoroquinolones, an analytical technique having the basic advantage of considerably
greater sensitivity as compared to spectrophotometry. The use of charge-transfer complex
formation involving chloranilic acid as a pi electrons acceptor might be desirable because
the reaction often provides reasonably high sensitivity, a wide dynamic range, stable
fluorescent species, and its applicability to drug analysis has been widely reported. The
191
Experimental
present work was carried out by exploiting this potential of fluoroquinolones and CL for
determination of fluoroquinolones in bulk and pharmaceutical formulations. The possible
charge-transfer complex formation of the drug with CL is given in figure 3.6.1.
Fluoroquinolones Chloranilic acid
Figure 3.6.1 The proposed reaction mechanism of fluoroquinolones with chloranilic acid
3.6.2 Preliminary investigation of the possibility of formation of charge-transfer

complex between fluoroquinolones and chloranilic acid for
spectrofluorometric determination of the drugs
Preliminary studies were conducted to investigate the possible formation of charge-

transfer (CT) complex between fluoroquinolones and CL acid. Initially high
concentration of fluoroquinolones (2.0 µg/mL) and CL acid (200 µg/mL) were mixed in a
volumetric flask and light purple colour appeared at once indicating successful formation
of the CT complex between the drugs and CL acid and subsequent spectrofluorometric
192
Experimental
determination of fluoroquinolones. Further studies were focused on optimization of

various parameters leading to the maximum possible enhancement of the fluorescence
intensity of the drug and are given below.
3.6.3 Optimization studies for the formation of charge-transfer complex between

fluoroquinolones and chloranilic acid
Instruments
Spectrofluorophotometer, (RF 5301, Shimadzu, Japan) with short arc Xenon lamp (UXL
– 155) was used for the measurement of fluorescence intensity, digital analytical balance
(Sartorius handy H51 Germany), clinical centrifuge (Model 800, China with maximum
speed 4000 rpm) and electric thermostatic water bath (MC 02810175 Yu Jia China) were
used during this investigation.
Reagents
All chemicals used were of high grade purity. Chloranilic acid (2,5-dichloro-3,6-
dihydroxy-p-bezoquinone), BDH Chemicals Ltd. Poole England, and methanol (Merck,
Darmstadt Germany) were used during this work. Standard reference levofloxacin,
moxifloxacin, gatifloxacin and ciprofloxacin were gifted by Libra Pharmaceutical
Industry pvt. Ltd., Peshawar, Pakistan and MKB Pharmaceutical Pvt. Limited Peshawar.
Commercial formulations of levofloxacin (Levoxin 500 mg/tab, Searle Pakistan Liminted
F-319, S.I.T.E., Karachi, Levotril 250 mg/tab Gray’s Pharmaceuticals Plot #442, St. #7,
I-9/2, Industrial area Islamabad, Leobac 500 mg/tab, Legacy Pharmaceutical (Pvt.) Ltd.
111-A, Industrial Estate, Hayatabad Peshawar Pakistan. Infusion Levflox 500mg/100
mL, Getz pharma (Pvt.) Ltd. Karachi Pakistan), moxifloxacin (tablet Moxibact 400
mg/tab and infusion Moxibact 400 mg/250 mL, manufactured by S. J. and G. Fazul
Ellahie (Pvt.) Ltd. Karachi Pakistan, under License Continental Pharmaceuticals Karachi
Pakistan, tablet Moxiget 400 mg/tab, manufactured by Getz Pharma (Pvt.) Ltd. Karachi
Pakistan and Megamox 0.5 % eye drops manufactured by Elko Organization (Pvt.) Ltd.
Karachi Pakistan and marketed by Sante (Pvt.) Limited Karachi Pakistan) and
ciprofloxacin ( Ciproxin 500mg/tab, Bayer Pakistan (Pvt.) Ltd. C-21, S.I.T.E. Karachi
193
Experimental
Pakistan, Mytil 500 mg/tab, Wilson’s Pharmaceuticals 387-88 I-9 Industrial Area
Islamabad Pakistan, Ciprax-500 500mg/tab, Gemone Pharmaceuticals (Pvt.) Ltd. Plot #
16/1 Phase IV, Industrial Estate, Hattar Pakistan.) were purchased from local medicine
store.
Chloranilic acid solution (500 µg/mL)
Pure solid chloranilic acid (0.025 g) was dissolved in 20 mL methanol and diluted up to
the mark in 50 mL volumetric flask with the same solvent. Working solutions of lower
concentration were prepared by dilution of the stock solution.
Standard fluoroquinolone solution (100 µg/mL)
Standard fluoroquinolone stock solution (100 µg/mL) was prepared by dissolving 0.01 g
of authentic standard fluoroquinolone in 10 mL of methanol with vigorous shaking and
finally diluted to 100 mL with distilled water. Working standard solutions of lower
concentrations were prepared by dilution of the stock solution with methanol.
Sample preparation (100 µg/mL)
Five tablets of each sample (Levoxin 500 mg/tab, Levotril 250 mg/tab, Levobac 500
mg/tab, Moxibact 400 mg/tab, Moxiget 400 mg/tab, Ciproxin 500mg/tab, Mytil 500
mg/tab, Ciprax-500 500mg/tab) were weighed separately to obtain average weight of one
tablet which was found to be 0.6308g, 0.4970g, 0.6439 g, 0.595 g, 0.610 g, 0.7767g,
0.7852 g and 0.7789 g for Levoxin, Levotril, Levobac, Moxibact, Moxiget, Ciproxin,
Mytil and Ciprax-500 respectively. The tablet forms of each brand were ground
separately. Sample of the powdered tablets equivalent to 0.01 g of the drug was
transferred to 100 mL beaker and dissolved in 10 mL methanol with vigorous shaking.
The solution was transferred to 100 mL volumetric flask and diluted up to the mark with
methanol to obtain 100 µg/mL sample solution in each case. These solutions were used as
stock sample solution throughout the work. For preparing stock solution of infusion
Levflox, Moxibact and eye drops Megamox, 100 µg/mL sample solutions was prepared
194
Experimental
by pipetting out sufficient volume directly and diluting with methanol in 100 mL
volumetric flask. Working sample solutions in the range of 1.0 – 5.0 µg/mL was prepared
by further dilution of the stock solution with same solvent.
Procedure
Solutions of the selected fluoroquinolones in the concentration range of 1.0 – 5.0 µg/mL
were taken in five separate 10 mL volumetric flask and chloranilic acid solution in the
concentration range of 10 – 200 µg/mL were added and made the volume up to mark
with methanol. A purple colour charge-transfer complex was formed between the
fluoroquinolones and chloranilic acid immediately. The fluorometric behaviour of the
drug with and without addition of chloranilic acid was measured to find wavelength for
maximum excitation (λex) and wavelength for maximum emission (λ em) of the drug and
CT complex. Excitation spectrum was obtained by scanning the analyte in the range of
260 – 350 nm and emission spectrum was obtained by scanning the analyte in the range
of 425 – 520 nm using spectrofluorometer and the results are shown in figure 3.6.2 –
3.6.5.
The effect of reaction medium on the fluorescence intensity of the complex was
investigated. Solutions of the selected fluoroquinolones (10.0 – 50.0 µg/mL) and solution
of chloranilic acid (10 – 200 µg/mL) were prepared in different solvents such as
methanol, ethanol, cyanomethane, n-hexane and distilled water. Aliquots of the selected
fluoroquinolones in the concentration range of 1.0 – 5.0 µg/mL were taken in 10 mL
volumetric flask and chloranilic acid solution in the concentration range of 10 – 200
µg/mL were added and made the volume up to mark with the same solvent. Each one of
these analyte solutions was scanned for emission wavelength in the respective range and
the result is shown in figure 3.6.6.
For optimization of concentration of CL acid, aliquots of CL acid (0.5 – 1.0 mL) in the
concentration range of 5 – 300 µg/mL were taken in 10 mL volumetric flasks and same
procedure was followed for obtaining the CT complex with selected fluoroquinolones.
Fluorescence intensity of each of these complex solutions was measured at their optimum
wavelengths and the results are shown in figures 3.6.7 – 3.6.10.
195
Experimental
For volume optimization of chloranilic acid, only volume of the reagent was varied in the
range of 0.25 – 2.0 mL and rest of the procedure was kept same. Fluorescence intensity
was measured at optimized wavelengths and the results are shown in figures 3.6.11.
To study the effect of heating temperature, aliquots of 0.5 mL of chloranilic acid solution
(50 µg/mL) were taken in six separate reaction flasks. To each flask 1.0 mL of the drug
(100 µg/mL) was added followed by addition of 5.0 mL methanol. The flasks were
heated on water bath for 10 minutes and heating temperature was varied in the range of
25 – 50 oC. The mixture was diluted to 10 mL in volumetric flask with methanol.
Fluorescence intensity of the solutions was measured at their optimized wavelengths.
Each experiment was conducted in triplicate and result is average of the triplicate
analysis. The results are shown in figure 3.6.12.
Stability of the CT complex between CL acid and the selected fluoroquinolones and the
subsequent stability of the proposed method was also investigated for two hour with 10
minutes interval and overnight reading was noted. The complex is quite stable and the
70
60
50
40
30
drug only
20
CT complex
10
0
250 300 350 400 450 500 550
Wavelength (nm)
Figure 3.6.2 Excitation and emission spectra of levofloxacin only ( ) and CT

complex (-----) with chloranilic acid
196
Experimental
140
120
100
80
Figure 3.6.3 Excitation and emission spectra of moxifloxacin only ( ) and CT

complex (-----) with chloranilic acid
60
60
40
50
20
400
250 300 350 400 450 500
Wavelength (nm)
Figure 3.6.4 Excitation and emission spectra of ciprofloxacin only ( ) and CT

complex30(-----) with chloranilic acid
20
197
10
Experimental
300
250
200
Figure 3.6.5 Excitation and emission spectra of gatifloxacin only ( ) and CT complex
(-----) with
150 chloranilic acid
70
100
60
50
40 methanol
30 cyanomethane
20 ethanol
50
10 aqueous
0 n-hexane
450 460 470 480 490 500 510 520
Wavelength (nm)
0
250 300 350 400 450 500
Wavelength (nm)
Figure 3.6.6 Fluorescence intensity of the CT complex as function of the medium
198
Experimental
70
60
50
40
Figure 3.6.7 Effect of concentration of CL acid on the spectrofluorometric

determination of levofloxacin
30
160
20
150
10
0
140
50 100 150 200 250
Conc. (µg/mL)

determination
130 of moxifloxacin
199
120
Experimental
370
360
350
340
Figure 3.6.9 Effect 330

of concentration of CL acid on the spectrofluorometric
determination of ciprofloxacin
320
310
270
300
250
290
280
230
0 50 100 150
Conc. (µg/mL)
orescence Intensity
200
210
Experimental

determination of gatifloxacin
400
300
Figure 3.6.11 Effect of volume of CL acid on the spectrofluorometric determination of

the selected
200 fluoroquinolones
380
100
0
0 0.5 1 1.5 2
Volume (mL) 201

scence Intensity
330
Experimental
Figure 3.6.12 Effect of temperature on fluorescence intenisty of the CT complex
100
80
60
40
20
0
0 10 20 30 40 50 60 70 80 90 100
Time (min)
Figure 3.6.13 Stability of fluorescence intenisty of the CT complex
3.6.4 Effect of concentration of fluoroquinolones on fluorescence intensity of the

CT complex
Instrument, reagent and solutions used were the same as mentioned before.
Procedure
Under the experimental conditions described above, standard calibration curve of CT

complex for levofloxacin, moxifloxacin, ciprofloxacin and gatifloxacin was constructed
in the concentration range of 0.06 – 3.2 µg/mL, 0.02 – 8.0 µg/mL and 0.02 – 10.0 µg/mL
respectively. The results are shown in figure 3.6.14 – 3.6.17.
replicates of analytes having lowest quantifiable concentration. Limit of detection and
limit of quantification were calculated using the following equations.
202
Experimental
All the optical parameters and statistical values calculated for the proposed method are
200
150
100
50
0
0 0.5 1 1.5 2 2.5 3 3.5
Conc. (µg/mL)
Figure 3.6.14 Effect of concentration of levofloxacin on the fluorometric behaviour of

CT complex
1000
800
600
Figure 3.6.15 Effect of concentration of moxifloxacin on the fluorometric behaviour of

CT complex
400
203
Experimental
400
300
Figure 3.6.16 Effect of concentration of ciprofloxacin on the fluorometric behaviour of

CT complex
200
600
100
500
4000
0 2 4 6 8
Conc. (µg/mL)
Figure 3.6.17 Effect of concentration of gatifloxacin on the fluorometric behaviour of

CT complex
300
200
204
Experimental
Table 3.6.1 Optical parameters for the spectrofluorometric determination of the selected fluoroquinolones by the
proposed method
Lev Moxi Cipro Gati
λex(nm) 285 292 330 285
λem (nm) 485 492 445 473
Concentration range (µg/mL) 0.06 – 3.2 0.02 – 8.0 0.02 – 10.0 0.02 – 10.0
LOD (µg/mL) 0.012 0.008 0.005 0.006
LOQ (µg/mL) 0.04 0.019 0.018 0.019
Regression equation (y) 58.37 X + 1.17 68.77 X + 0.95 71.04 X + 0.93 70.31 X + 0.97
Slope (b) 58.37 68.77 71.04 70.31
Intercept (a) 1.17 0.95 0.93 0.97
Correlation coefficient 1 0.9929 0.9952 0.9975
Relative standard deviation (%) 7.79 6.20 6.90 6.36
205
Experimental
3.6.5 Effect of common interferers on determination of the selected

fluoroquinolones using the proposed method
Procedure
preparations containing the selected fluoroquinolones, interferences effect of common
excipients added to commercial formulations was investigated. For this purpose a known
concentration (1.0 µg/mL) of each selected standard fluoroquinolones was analyzed at
optimum conditions. Then the same concentration of each selected standard
fluoroquinolones was taken in separate volumetric flasks and different concentrations of
the excipients in the ratio of 1:5, 1:10 and 1:50 were added to it. Optimized amount of CL
acid for each drug was added to it and CT complex was prepared in the same way as
mentioned before. Fluorescence intensity of the complex was measured at respective
wavelengths and percent recovery of the analyte was calculated. Each experiment was
performed in triplicate and mean fluorescence intensity was calculated. The results are
given in table 3.6.2 and are shown in figure 3.6.18.
205
Experimental
Table 3.6.2 Interferences effect of common excipients on % recovery of the

analyte (1.0 µg/mL) using the proposed method
Excipient Amount Ratio Recovery

added (%) ± RSD
(µg/mL)
5.0 5.0 98.98 ± 0.35
Glucose 10 10 97.93 ± 0.26
50 50 99.92 ± 0.24
5.0 5.0 99.97 ± 0.31
Fructose 10 10 102.07 ± 0.29
50 50 100.03 ± 0.21
5.0 5.0 102.02 ± 0.25
Sucrose 10 10 100.01 ± 0.34
50 50 98.89 ± 0.22
5.0 5.0 102.10 ± 0.28
Sorbitol 10 10 100.05 ± 0.20
50 50 97.95 ± 1.49
5.0 5.0 97.87 ± 2.22
Lactose 10 10 102.02 ± 3.06
50 50 99.95 ± 3.43
5.0 5.0 100.03 ± 2.24
Starch 10 10 98.94 ± 2.73
50 50 98.94 ± 2.26
5.0 5.0 97.95 ± 3.63
Talc 10 10 100.08 ± 3.81
50 50 96.92 ± 3.14
5.0 5.0 96.97 ± 2.57
Magnesium stearate 10 10 97.98 ± 3.12
50 50 98.99 ± 2.55
5.0 5.0 100.03 ± 2.43
Sodium citrate 10 10 99.96 ± 2.38
50 50 99.91 ± 2.12
5.0 5.0 100.04 ± 2.53
Methyl cellulose 10 10 100.01 ± 2.33
50 50 99.98 ± 3.41
206
Experimental
105
100
Percent recovery
95
90
l c
FQ cose tose rose ito tose arch Tal ate ate lose
u ruc uc orb ac St r tr u
l
G F S S L S tea . Ci ell
g. od . C
M S M
1:00 1:05 1:10 1:50
Figure 3.6.18 Interferences effect of common excipients on % recovery of the analyte

(1.0 µg/mL) using the proposed method
Effect of some common cations typically found in human urine and blood samples on
fluorescence intensity of the CT complex using the selected fluoroquinolones was also
investigated. For this purpose a known concentration (1.0 µg/mL) of the selected
standard fluoroquinolones was analyzed. Then the same concentration of the standard
drugs and different concentration of the possible cations in different concentration ratio
(1:50, 1:100 and 1:500) were added to a series of volumetric flasks (10 mL) followed by
the addition of optimized amount of CL acid and diluted up to the mark with methanol.
The fluorescence intensity of each solution was measured and the results are given in
table 3.6.3 and shown in figure 3.6.19. Each experiment was performed in triplicates and
mean percent recovery was obtained.
207
Experimental
Table 3.6.3 Effect of common cations found in human plasma and urine samples on
determination of the selected fluoroquinolones (1.0 µg/mL)
Amount added Ratio Recovery (%) ±

Cation
(µg/mL) RSD
50 50 101.63 ± 2.15
Na+ 100 100 101.32 ± 1.55
500 500 99.91 ± 2.22
50 50 100.11 ± 1.98
K+ 100 100 101.78 ± 1.62
500 500 99.03 ± 2.55
50 50 97.15 ± 1.19
NH4+ 100 100 100.43 ± 2.92
500 500 101.11 ± 1.89
50 50 98.49 ± 2.02
Ca2+ 100 100 100.32 ± 1.99
300 300 96.66 ± 2.22
50 50 100.54 ± 1.83
Mg2+ 100 100 98.09 ± 1.36
300 300 98.73 ± 1.88
50 50 99.36 ± 2.32
Zn2+ 100 100 97.77 ± 1.74
500 500 101.73 ± 2.55
50 50 100.36 ± 1.53
Cu2+ 100 100 101.58 ± 1.43
500 500 99.62 ± 2.09
50 50 101.68 ± 1.43
Fe2+ 100 100 98.71 ± 1.22
500 500 99.28 ± 1.90
5 5 100.85 ± 1.89
Al3+ 10 10 101.39 ± 1.35
50 50 100.46 ± 1.87
5 5 97.83 ± 1.39
Fe3+ 10 10 98.38 ± 1.76
15 15 100.19 ± 1.99
208
Experimental
Figure 3.6.19 Effect of common cations found in human plasma and urine samples on
determination of the selected fluoroquinolones (1.0 µg/mL)
The effect of some common compounds typically found in human urine samples on
fluorescence intensity of the CT complex of the selected fluoroquinolones with CL acid
was also investigated. For this purpose a known concentration (0.5 µg/mL) of the
selected standard fluoroquinolones was analyzed and then the same concentration of the
standard drugs and different concentration of the commonly occurring compounds in
human urine like urea, uric acid, lactic acid, creatinine and citric acid in different
concentration ratio (1:50, 1:100 and 1:500) were added to a series of volumetric flasks
(10.0 mL) followed by the addition of optimized amount of CL acid and diluted up to the
mark with methanol. The fluorescence intensity of each solution was measured and the
results are given in table 3.6.4 and shown in figure 3.6.20. Each experiment was
performed in triplicate and mean percent recovery was obtained.
209
Experimental
Table 3.6.4 Effect of common compounds occurring in human urine samples on

determination of levofloxacin (0.5 µg/mL)
Compound Added (µg/mL) Ratio Recovery (%) ±

RSD
5 10 100.19 ± 0.553
Urea 25 50 100.14 ± 0.439
50 100 99.82 ± 0.773
5 10 99.68 ± 0.688
Uric Acid 25 50 100.31 ± 0.498
50 100 100.55 ± 0.699
5 10 99.32 ± 0.821
Citric acid 25 50 99.39 ± 0.783
50 100 99.11 ± 0.611
5 10 100.43 ± 0.552
Lactic acid 25 50 99.97 ± 0.712
50 100 100.48 ± 0.696
5 10 99.88 ± 0.580
Creatinine 25 50 100.21 ± 0.668
50 100 98.99 ± 0.598
Figure 3.6.20 Effect of common compounds occurring in human urine samples on

determination of levofloxacin (0.5 µg/mL)
The effect of the presence of co-administered drugs such as aspirin, mefenamic acid,
paracetamol, vitamin C, metronidazole, sodium diclofenac, brufen and magnesium
210
Experimental
tricilicate and famotidine on fluorescence intensity of the CT complex of the selected

fluoroquinolones with CL acid was also investigated. For this purpose a known
concentration (0.5 µg/mL) of the selected standard fluoroquinolones was analyzed and
then the same concentration of the standard drugs and different concentration of the co-
administered drugs in different concentration ratio (1:2, 1:5 and 1:10) were added to a
series of volumetric flasks (10.0 mL) and rest of the procedure was kept the same. The
fluorescence intensity of each solution was measured. Each experiment was performed in
triplicate and mean percent recovery was obtained and the results are given in table 3.6.5
and shown in figure 3.6.21.
211
Experimental
Table 3.6.5 Effect of co-administered drugs on determination the selected FQ using

Compound Added Ratio Recovery (%)

(µg/mL) ± RSD
1.0 2 99.87 ± 0.622
Aspirin 2.5 5 99.79 ± 0.701
5.0 10 100.09 ± 0.717
1.0 2 99.88 ± 0.492
Mefenamic acid 2.5 5 100.19 ± 0.616
5.0 10 100.43 ± 0.509
1.0 2 100.29 ± 0.489
Paracetamol 2.5 5 99.99 ± 0.682
5.0 10 100.33 ± 0.739
1.0 2 100.09 ± 0.326
Vitamin C 2.5 5 99.79 ± 0.636
5.0 10 99.91 ± 0.703
1.0 2 99.75 ± 0.329
Metronidazole 2.5 5 100.82 ± 0.453
5.0 10 99.87 ± 0.746
1.0 2 100.63 ± 0.771
Sodium Diclofenac 2.5 5 101.02 ± 0.883
5.0 10 99.33 ± 0.644
1.0 2 99.29 ± 0.647
Brufen 2.5 5 100.73 ± 0.730
5.0 10 100.03 ± 0.778
1.0 2 99.59 ± 0.490
Magnesium Tricilicate 2.5 5 99.77 ± 0.641
5.0 10 100.38 ± 0.587
1.0 2 98.44 ± 0.828
Famotidine 2.5 5 97.30 ± 0.703
5.0 10 97.21 ± 0.880
212
Experimental
110
105
100
Figure 3.6.21 Effect of co-administered drugs on determination the selected FQ using
Percent recovery
3.6.6 Application of the proposed method for the analysis of the selected drugs in
95
A. Precision and accuracy of the investigated method
90
before.
Procedure
Precision and accuracy 85

of the investigated method was determined by analysis of ten
separate sample solutions of FQthe commercial
pi
ri n id
formulations
ac
ol
m ((Levoxin
C le
in 500 mg/tab,
zo ac u
As ta m a fen Br
ic c e ita i d l o
am
Levotril 250 mg/tab, Levobac 500 mg/tab, Levflox 500 on
ra mg/100 VmL, Moxibact 400. Dic
fe en Pa etr d
M M So
mg/tab, Moxiget 400 mg/tab, Moxibact 400mg/250 mL, Megamox 0.5 %, Ciproxin 500
mg/tab, Mytil 500 mg/tab, Ciprax-500 500mg/tab) at three different concentration levels
(0.1, 0.2 and 0.4 µg/mL). Calibration curve was constructed by the same procedure as
mentioned before and known concentrations of these sample solution were then analyzed
by the proposed method in triplicate. The results are shown in table 3.6.6.
213
Experimental
Table 3.6.6 Evaluation of accuracy and precision of the proposed method for the
determination of selected fluoroquinolones
Sample Amount taken Amount found Recovery ±

0.100 0.099 99.0 ± 1.66
Levoxin (500 mg/tab) 0.200 0.198 99.0 ± 1.86
0.400 0.409 102.3 ± 1.52
0.100 0.096 96.0 ± 2.12
Levotril (250 mg/tab) 0.200 0.202 101.0 ± 2.13
0.400 0.396 96.6 ± 2.72
0.100 0.103 91.0 ± 1.18
Levobac (500 mg/tab) 0.200 0.197 95.0 ± 1.32
0.400 0.403 96.6 ± 0.86
0.100 0.102 102.0 ± 1.56
Levflox (500 mg/100 mL) 0.200 0.201 100.5 ± 1.33
0.400 0.401 100.3 ± 1.69
0.100 0.095 95.0 ± 2.17
Moxibact (400 mg/tab) 0.200 0.197 98.5 ± 1.93
0.400 0.398 99.5 ± 1.92
0.100 0.096 96.0 ± 1.98
Moxiget (400 mg/tab) 0.200 0.204 102.0 ± 1.92
0.400 0.399 99.8 ± 1.96
0.100 0.102 102.0 ± 2.06
Moxibact (400 mg/250 0.200 0.199 99.5 ± 1.89
mL)
0.400 0.403 100.8 ± 1.92
0.100 0.099 99.0 ± 1.15
Megamox (0.5 %) 0.200 0.201 100.5 ± 1.13
0.400 0.403 100.8 ± 1.22
0.100 0.101 101.0 ± 2.08
Ciproxin (500 mg/tab) 0.200 0.197 98.5 ± 2.12
0.400 0.397 99.3 ± 2.06
0.100 0.097 97.0 ± 2.61
Mytil (500 mg/tab) 0.200 0.195 97.5 ± 1.81
0.400 0.395 98.8 ± 2.59
0.100 0.095 95.0 ± 2.10
Ciprax-500 (500 mg/tab) 0.200 0.196 98.0 ± 2.11
0.400 0.397 99.3 ± 1.02
214
Experimental
B. Percent recovery of the selected drugs from commercial formulations

(Standard addition method)
before.
Procedure
For determination of percent recovery of standard selected fluoroquinolones from

commercial formulation, 0.1, 0.2 and 0.4 µg/mL standard drugs solution was added to
preanalyzed 0.2 µg/mL commercial sample of the selected fluoroquinolones and
fluorescence intensity was measured by following the same procedure as mentioned
before. The experiments were performed in triplicate and the mean percent recovery was
calculated using the following formula. The results are given in table 3.6.7.
215
Experimental
Table 3.6.7 Evaluation of recovery (%) of the selected drugs from commercial
Sample Sample Added Found Recovery

(µg/mL) (µg/mL) (µg/mL) (%) ± RSD
0.100 0.297 97.0 ± 2.09
Levoxin (500 mg/tab) 0.2 0.200 0.399 99.5 ± 2.11
0.400 0.603 100.8 ± 2.16
0.100 0.294 94.0 ± 1.85
Levotril (250 mg/tab) 0.2 0.200 0.406 103.0 ± 1.79
0.400 0.596 99.0 ± 1.91
0.100 0.302 102.0 ± 2.22
Levobac (500 mg/tab) 0.2 0.200 0.397 98.5 ± 2.19
0.400 0.603 100.8 ± 2.26
0.100 0.302 102.0 ± 1.73
Levflox (500 mg/100 mL) 0.2 0.200 0.407 103.5 ± 1.69
0.400 0.606 101.5 ± 1.69
0.100 0.297 97.0 ± 1.53
Moxibact (400 mg/tab) 0.2 0.200 0.395 97.5 ± 1.47
0.400 0.598 99.5 ± 1.55
0.100 0.296 96.0 ± 2.68
Moxiget (400 mg/tab) 0.2 0.200 0.404 101.0 ± 2.63
0.400 0.599 99.8 ± 2.77
0.100 0.302 102.0 ± 1.83
Moxibact (400 mg/250 mL) 0.2 0.200 0.399 99.5 ± 1.87
0.400 0.603 100.8 ± 1.88
0.100 0.299 99.0 ± 2.36
Megamox (0.5 %) 0.2 0.200 0.404 102.0 ± 2.44
0.400 0.601 100.3 ± 2.52
0.100 0.303 103.0 ± 1.17
Ciproxin (500 mg/tab) 0.2 0.200 0.397 98.5 ± 1.28
0.400 0.595 98.8 ± 1.11
0.100 0.295 95.0 ± 1.68
Mytil (500 mg/tab) 0.2 0.200 0.395 97.5 ± 1.73
0.400 0.596 99.0 ± 1.44
0.100 0.294 94.0 ± 1.73
Ciprax-500 (500mg/tab) 0.2 0.200 0.399 99.5 ± 1.66
0.400 0.594 98.8 ± 1.79
216
Experimental
before.
Procedure
Different commercial formulations were purchased from local market. The samples of the
powdered tablets (0.013g, 0.020g, 0.013g, 0.012g, 0.012g, 0.016g, 0.016g and 0.016g for,
Levoxin, Levotril, Levobac, Moxibact, Moxiget, Ciproxin, Mytil and Ciprax-500
respectively and 6.25 mL for infusion Moxibact and 2 mL each for Levflox and
Megamox eye drops), equivalent to 0.01g of the selected fluoroquinolones were
transferred to 100 mL beaker and 10 mL methanol was added to it and shaken vigorously
to dissolve all the contents. The contents were transferred to 100 mL volumetric flask and
made the volume up to the mark with methanol to obtain 100 µg/mL sample solution.
The solution was filtered and working sample solution (10.0 µg/mL) was prepared by
dilution with same solvent. Known volumes (1.0 mL of 10.0 µg/mL) of this sample
solution were then analyzed by the proposed method in triplicate. The results are given in
table 3.6.8.
217
Experimental
Table 3.6.8 Determination of active ingredients in the commercial formulations using

the proposed method
Brand name Active ingredient t test value

Tab. Levoxin (mg/tab) 500 497.18 ± 0.25 0.15
Tab. Levotril (mg/tab) 250 251.86 ± 0.19 0.11
Tab. Levobac (mg/tab) 500 492.77 ± 0.13 0.45
Inf. Levflox (mg/100 mL) 500 501.32 ± 0.25 0.19
Tab. Moxibact (mg/tab) 400 399.13 ± 0.19 0.21
Tab. Moxiget (mg/tab) 400 397.92 ± 0.13 0.29
Inf. Moxibact (mg/250 mL) 400 400.25 ± 0.25 0.11
Megamox eye drops (mg/100 mL) 500 500.11 ± 0.19 0.17
Tab. Ciproxin (mg/tab) 500 497.33 ± 0.13 0.33
Tab. Mytil (mg/tab) 500 495.99 ± 0.25 0.51
Tab. Ciprax-500 (mg/tab) 500 494.47 ± 0.19 0.49
D. Validation of the proposed method in spiked biological samples
a. Spiked urine sample
Procedure
Urine samples from three healthy volunteers at normal conditions were taken and diluted
ten times with distilled water (5.0 mL of the urine were diluted to 50 mL with distilled
water). Levofloxacin solution (1.0 mL of 100 µg/mL) was added to 1.0 mL urine sample
and diluted to 5.0 mL with methanol. The spiked sample was centrifuged at 3000 rpm for
15 minutes and 5.0 µg/mL working spiked urine sample solution was prepared by
pipetting out 2.5 mL of the clear supernatant portion and diluting to 10 mL with
methanol. Spiked urine sample solution (5.0 µg/mL) in the range of 1 – 3 mL was
transferred to 10 mL volumetric flask. To each flask, 0.5 mL of 200 µg/mL chloranilic
acid solution was added and made the volume up to mark with pure methanol to give
218
Experimental
final concentration of the drug in the range of 0.5 - 1.5 µg/mL. Fluorescence intensity of
the solution was measured using 285 nm as excitation wavelength and 485 nm as
emission wavelength. The results are given in table 3.6.9.
Table 3.6.9 Spectrofluorometric determination of levofloxacin in spiked urine by the

proposed method
Sample Spiked urine

0.50 0.50 100.0 ± 1.73
Urine 1.00 1.00 100.0 ± 1.64
1.50 1.50 100.0 ± 1.56
b. Spiked plasma sample
Procedure
In case of plasma, 4.0 mL was spiked with 1.0 mL of the standard levofloxacin (500
µg/mL) and 5.0 mL of cyanomethane was added for deprotination. The precipitated
protein was separated by centrifugation at 3500 rpm for 20 minutes. Appropriate volumes
of the supernatant spiked plasma was transferred to 25 mL volumetric flask and diluted
with methanol to give nominal concentration of 5.0 µg/mL of the drug and 1 - 3 mL of
this solution was transferred to 10 mL volumetric flask. To each flask, 0.5 mL of 200
µg/mL chloranilic acid solution was added and made the volume up to mark with pure
methanol to give final concentration of the drug in the range of 0.5 - 1.5 µg/mL.
Fluorescence intensity of the solution was measured using optimized conditions and
concentration of the drug was evaluated from a calibration curve constructed separately
from standards containing equivalent amount of cyanomethane. Each experiment was
performed in triplicate. The average percent recovery was calculated and the results are
219
Experimental
Table 3.6.10 Spectrofluorimetric determination of levofloxacin in spiked plasma by the

proposed method

0.50 0.50 100.0 ± 1.26
Plasma 1.00 1.01 101.0 ± 1.36
1.50 1.50 100.0 ± 1.25
A simple, fast and accurate spectrofluorometric method for determination of the selected
fluoroquinolones (levofloxacin, moxifloxacin, ciprofloxacin and gatifloxacin) in bulk,
pharmaceutical formulation and biological samples has been developed and validated.
The method is based on fluorescence enhancement phenomenon by reaction of the
fluoroquinolones with chloranilic acid. This enhanced signal extended the linear range on
both side and made the measurement of even trace quantity of the drugs possible.
To establish the most favorable conditions for the enhanced fluorescence intensity of the
selected fluoroquinolones, all variables affecting the reaction such as concentration and
volume of the complexation agent and reaction temperature were carefully studied and
optimized.
Fluorimetric behaviour of the CT complex is solvent dependent. The selection of

appropriate solvent plays a vital role in the sensitivity of the method. Effect of different
solvent such as methanol, cyanomethane, ethanol, distilled water and n-hexane on
fluorimetric behaviour of CT complex between the selected FQs and CL acid was
investigated. Methanol was found to be the best solvent (figure 3.6.6) in terms of
sensitivity, environmental aspect and cost of the method.
Concentration of CL acid solution affects the reaction markedly. Its effect was studied in
the concentration range of 100 – 300 µg/mL, 5.0 – 25 µg/mL, 25 – 200 µg/mL and 30 –
70 µg/mL for levofloxacin, moxifloxacin, ciprofloxacin and gatifloxacin respectively. It
was observed that with increase in concentration of CL acid up to 200 µg/mL, 15 µg/mL
220
Experimental
and 50 µg/mL for levofloxacin, moxifloxacin, ciprofloxacin and gatifloxacin

respectively, FI intensity of the complex increases and then decrease with further increase
in concentration of the reagent (figure 3.6.7 – 10). Therefore, these concentrations were
used in further analyses of the respective drugs. The effect of volume of optimized
concentration of CL acid for each drug on the fluorescence intensity of the analyte was
also investigated in the range of 0.25 – 2.0 mL and 0.5 mL was found as the optimum
volume for levofloxacin and ciprofloxacin and 1.0 mL for moxifloxacin and gatifloxacin
(figure 3.6.11).
Increase in temperature has negative effect on the fluorescence intensity of the analyte.
Generally, it happens due to changes in structure of molecules and emission less
relaxation of the excited states. Good results were obtained at room temperature (figure
3.6.12) and thus making the process easier; further analyses were carried out at room
temperature.
Stability of the CT complex between CL acid and the selected fluoroquinolones and the
subsequent stability of the proposed method was also investigated for two hour with 10
minutes interval and also overnight reading was noted. The complex is quite stable
(figure 3.6.13) and gives a consistent signal even after over a night stay.
Under the optimum experimental conditions of the proposed method, a linear response
between the fluorescence intensity and concentration of the selected FQs was observed in
the concentration range of 0.06 – 3.2 µg/mL, 0.02 – 8.0 µg/mL and 0.02 – 10.0 µg/mL
for levofloxacin, moxifloxacin, ciprofloxacin and gatifloxacin respectively (figure 3.6.14
– 17). The linearity of calibration curves was proved by the high value of correlation
coefficient. The linear regression equations, slopes, intercepts, correlation coefficients,
standard deviation and relative standard deviation of the response factors are given in
table 3.6.1. The limit of detection (LOD) was found to be 0.012 µg/mL, 0.008 µg/mL,
0.005 µg/mL and 0.006 µg/mL for levofloxacin, moxifloxacin, ciprofloxacin and
gatifloxacin respectively. Limit of quantification (LOQ) was found to be 0.04 µg/mL,
221
Experimental
0.019 µg/mL, 0.018 µg/mL and 0.019 µg/mL for levofloxacin, moxifloxacin,
ciprofloxacin and gatifloxacin respectively.
Application
For evaluation of the selectivity of the proposed method for the analysis of
pharmaceutical preparations containing the selected FQs, the interferences effect of
various pharmaceutical additives such as glucose, fructose, lactose, sucrose, sorbitol,
magnesium stearate, sodium citrate, methyl cellulose, talc and starch was studied.
Solutions containing moxifloxacin and one of the excipients taken separately in
concentrations five, ten and fifty times greater than that of the selected FQs were
analyzed by the proposed method. A level of interference was considered to be
acceptable if the error was not higher than 3% relative to the expected value of drug. The
results indicated that there was no significant interferences effect by the excipients
studied on the fluorimetric behaviour of the drugs (table 3.6.2 and figure 3.6.18).
Fluorimetric behaviour of the CT complex of the selected FQs and CL acid was also
investigated in the presence of some common cations typically found in urine and blood
plasma. The fluorescence intensity of the complex was found free from the interferences
effect of these ions except Ca2+ and Mg2+ which decrease fluorescence behaviour of the
selected FQs at concentration level greater than 300 µg/mL (table 3.6.3 and figure
3.6.19). This decrease in fluorescence intensity is mainly due to complex formation of the
reagent with these cations at higher concentration. The major constituents of urine such
as urea, uric acid etc. also do not have any interference effect on the method.
Fluorescence intensity of the CT complex of the selected FQs and CL acid was measured
in the presence of compounds occurring in urine and found good percent recovery (table
3.6.4 and figure 3.6.20). Mostly antibiotics are not given alone, some antipyretic,
analgesic or vitamins are also given along with the antibiotics to the patient and thus
effect of the presence of these co-administered drugs on the determination of the selected
FQs was investigated. It was observed that the studied co-administered drugs do not
affect fluorescence behaviour and therefore, good percent recovery was obtained (table
3.6.5 and figure 3.6.21).
222
Experimental
The precision and accuracy of the proposed method was checked by determining the
selected FQs in replicate for each concentration within the linear range of concentration.
The results are summarized in table 3.6.6. The relative standard deviation (RSD)
considered being very satisfactory and the recovery test was found to be in the range of
95.0 ± 2.17 % to 102.3 ± 1.52 % for different brands of the selected FQs which indicates
a good accuracy.
The validity of the proposed method in dosage form of eleven different brands was tested
for possible interference using standard addition method. The average percent recoveries
obtained were in the range of 94.0 ± 1.73 % to 103.5 ± 1.69 % (table 3.6.7) which shows
a good accuracy of the proposed method for determination of the selected FQs in
commercial pharmaceutical preparations. The proposed method has been successfully
applied for the determination of the drugs in commercial preparations (tablets, infusion
and eye drops). For all the formulations examined, the results obtained for
pharmaceutical dosage forms (table 3.6.8) were in good agreement with the label claimed
value.
Good sensitivity obtained by the proposed method indicated the possibility of its
application for the quantification of the selected FQs in urine and plasma samples. FQs
are orally or parentally administered at doses of 400 mg once a day, which results in
plasma level of concentration of about 0.3 to 3.0 μg/mL. For accurate determination of
these drugs in biological samples, standard addition method was used for quantitative
determination of the selected FQs in urine and plasma samples. The results of the
recovery from urine and plasma samples by the proposed method are given in table 3.6.9
and 3.6.10. The results obtained were satisfactorily precise, accurate and the method can
be used for determination of the drugs concentration in biological samples in clinical
analysis.
223
Conclusions
CONCLUSIONS
Aim of the present research work was to develop fast, inexpensive and simpler
spectrofluorimetric and spectrophotometric methods for determination of selected
fluoroquinolones widely available in the market and manufactured by the local units.
Three different spectrophotometric methods were developed for determination of
sparfloxacin and three spectrofluorimetric methods were developed for determination of
ciprofloxacin, levofloxacin, gatifloxacin and moxifloxacin and were applied for the
analyses of the selected fluoroquinolones in commercial formulations and biological
samples.
The first spectrophotometric method is for determination of sparfloxacin in

pharmaceutical preparations based on oxidation of the drug using ammonium
monovanadate in acidic media. The proposed method was successfully applied for the
determination of sparfloxacin in different pharmaceutical formulations (tablets) and
spiked synthetic urine sample. The method is simple, precise, and accurate and was
statistically evaluated. As compared to HPLC methods, the reagents used in the proposed
method are readily available and the procedure did not involve any tedious sample
preparation.
The second spectrophotometric method is for quantification of sparfloxacin in

pharmaceutical dosages and biological samples. The method is based on the condensation
reaction between sparfloxacin and DMAB in acidic medium at room temperature to
produce a yellow coloured product. The proposed method is accurate, sensitive, precise,
rapid, and suitable for the determination of sparfloxacin in pure form and can be
satisfactorily applied to the quality control analysis of pharmaceutical preparations
containing the drug and biological samples. As compared to HPLC literature method, the
reagents used in the proposed spectrophotometric method are inexpensive and readily
available. The method is simple and does not require any critical reaction conditions or
sample preparation. The method is free from interferences due to common excipients.
The method is comparable in terms of sensitivity and reproducibility with literature
methods.
224
Conclusions
The third proposed spectrophotometric method is based on oxidation of sparfloxacin

using cerium (IV) in acidic medium with subsequent measurement of decrease in
absorbance of cerium (IV) at 315 nm. Under the optimum conditions, Beer’s law was
obeyed in the range 0.02 – 0.2 µg/mL. The method can be suitably adopted for routine
analysis to check the labeled contents in their commercial formulations. The proposed
method is simple and readily adaptable in the quality control laboratories.
The fourth method is a spectrofluorimetric method proposed for determination of

sparfloxacin and is based on oxidation of sparfloxacin by cerium (IV) in acidic medium
with subsequent measurement of fluorescence intensity of the product cerium (III) at
352 nm using excitation wavelength of 250 nm. Under the optimum conditions, linear
range of the method was found to be 0.02 – 0.1 µg/mL. The method was successfully
applied for the analysis of the investigated drug in pure and pharmaceutical formulations
with good accuracy and precision. The percentage recovery ranged from 93.0 ± 2.6 % –
102.2 ± 2.3 %. Interference effects of various related compounds have been investigated
on the determination of sparfloxacin and the method was found free of interferences.
The fifth proposed method is micellar-enhanced spectrofluorimetric determination of

moxifloxacin in pharmaceutical formulations and biological samples. Experimental
parameters that effect fluorescence intensity of the drug were investigated and optimized.
A linear relationship was found between fluorescence intensity and moxifloxacin
concentration in the range of 1.25 ng/mL to 3200 ng/mL containing 0.03M sodium
dodecyl sulphate using λex 294 nm and λem 494 nm with a good correlation coefficient.
The proposed spectrofluorimetric method is fast, simple and accurate. The method can be
used for routine analysis of moxifloxacin in pharmaceutical formulations as well as for
analysis in urine and plasma samples. The proposed method is a good alternative to the
conventional determination of moxifloxacin through HPLC methods with good
sensitivity.
The last proposed spectrofluorimetric method is for determination of the selected range of
fluoroquinolones and is based on monitoring the fluorescence intensity of the charged
transfer (CT) complex formed between chloranilic acid and ciprofloxacin, levofloxacin,
gatifloxacin and moxifloxacin at 445 nm, 485 nm, 473 and 492 nm using an excitation
225
Conclusions
wavelength of 330 nm, 285 nm, 285 nm and 292 nm respectively. The proposed method
is simple, fast, sensitive and accurate. The method was applied for determination of the
drugs in tablets, infusion and eye drops formulations with mean percent recovery of
101.58 with high accuracy. The results obtained for recovery, precision and accuracy
show that the proposed method could be successfully used for identification,
determination and quantification of the selected fluoroquinolones in commercial
formulations and biological samples. Common excipients used as additive in
pharmaceutical preparations, some common cations and compounds present in urine and
plasma and co-administered analgesic, vitamins and other drugs do not interfere with the
method.
226

Inayatullah Thesis Chemistry

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Inayatullah Thesis Chemistry

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SPECTROFLUORIMETRIC AND

SPECTROPHOTOMETRIC METHODS FOR

INSTITUTE OF CHEMICAL SCIENCES

SUBMITTED TO THE UNIVERSITY OF PESHAWAR IN

INSTITUTE OF CHEMICAL SCIENCES

Prof. Syed Ghawas. I am indebted to him for opening my

mind and touching my heart.

I thank her as the earth thanks the sun.

Your steadfastness made that which follows, real.

May the peace and blessings of Allah be upon our Prophet,

First and foremost, I am deeply indebted to my teacher and my

Words cannot express the nature and extent of my deepest

I am really short of words to express my gratitude to my uncle Dr.

My thanks are due to the Higher Education Commission of Pakistan,

Mention must be made of my worthy professors and teachers at the

I give my estranged thanks to Imdadullah Muhammadzai Ph.D.,

Very special thanks and my deep gratitude go to Dr. Muhammad

I am grateful to my senior scholar colleagues for providing a

I take this opportunity to acknowledge with thanks the help and

Where would I be without my family? I want to express deepest

S. No. TITLE Page No.

I LIST OF FIGURES i-v

II LIST OF TABLES vi-ix

III ABBREVIATIONS x-xiii

1.1.1 Physical Characteristics 15

1.1.2 Dosage and mode of administration 16

1.1.3 Over dosage 17

1.2.1 Physical Characteristics 17

1.3.1 Physical Characteristics 19

1.3.2 Dosage and mode of administration 20

1.4.1 Physical Characteristics 21

1.4.2 Dosage and mode of administration 22

1.4.3 Over dosage 22

1.5.1 Physical Characteristics 23

1.5.2 Moxifloxacin Dosing Guidelines 24

1.5.3 Over dosage 24

CHAPTER-2: LITERATURE REVIEW

CHAPTER-3: EXPERIMENTAL/RESULTS AND DISCUSSION

3.1.0 Investigation of spectrophotometric method for

3.1.1 Method development strategy for the spectrophotometric

3.1.2 Preliminary investigation of the possibility of oxidation of 91

3.1.3 Optimization studies for spectrophotometric determination of

3.1.4 Verification of Beer’s law for determination of sparfloxacin by

3.1.5 Application of the investigated method for the analysis of

3.1.6 Validation of the proposed method for biological samples

3.1.7 Results and discussion 105

3.2.0 Quantification of sparfloxacin in pharmaceutical dosages and

3.2.1 Method development strategy for the spectrophotometric

3.2.2 Preliminary investigation of the possibility of condensation

3.2.3 Optimization studies for spectrophotometric determination of

3.2.4 Verification of Beer’s law for determination of sparfloxacin by

3.2.5 Effect of common excipients found in commercial formulations

3.2.7 Validation of the proposed method in spiked biological samples 124

3.2.8 Determination of sparfloxacin by reference method (HPLC

3.2.9 Results and discussion 127

3.3.0 Indirect spectrophotometric method for determination of

3.3.1 Method development strategy for the spectrophotometric

3.3.2 Preliminary investigation of the possibility of oxidation of

3.3.3 Investigation of suitable parameter for the determination of

3.3.4 Verification of Beer’s law and determination of limit of detection

3.3.5 Effect of common excipients found in commercial formulations

3.3.6 Application of the investigated method for the analysis of

3.3.7 Percent recovery of sparfloxacin from commercial formulations

3.3.8 Results and discussion 144