“ANALYTICAL METHOD DEVELOPMENT AND VALIDATION FOR THE
DETERMINATION OF HYOSCINE BUTYLBROMIDE AND OFLOXACIN
HYDROCHLORIDE IN BULK AND MARKETED FORMULATIONS”
MASTER OF PHARMACY DISSERTATION PROTOCOL
SUBMITTED TO THE
RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES KARNATAKA,
BANGALORE
BY
KERKAR SANAM SAULO
M.PHARM – I
Under The Guidance of
Dr. E.V.S. SUBRAHMANYAM. M.PHARM.Ph.D
DEPARTMENT OF QUALITY ASSURANCE,
SRINIVAS COLLEGE OF PHARMACY, MANGALORE – 574143
2012-2014
RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES
BANGALORE, KARNATAKA
ANNEXURE – II
REGISTRATION OF SUBJECT FOR DISSERTATION
KERKAR SANAM SAULO
1.0
NAME AND ADDRESS OF
I YEAR M. PHARM,
THE CANDIDATE
DEPARTMENT OF Q.A.,
SRINIVAS COLLEGE OF PHARMACY,
VALACHIL,POST PARENGIPETE,
MANGALORE -574143
2.0
3.0
NAME
OF
THE
SRINIVAS COLLEGE OF PHARMACY,
INSTITUTION
VALACHIL, MANGALORE.
COURSE OF STUDY &
MASTER OF PHARMACY
SUBJECT
(QUALITY ASSURANCE)
4.0
DATE OF ADMISSION
5.0
TITLE OF THE TOPIC:
26TH MAY 2012
“ANALYTICAL METHOD DEVELOPMENT AND VALIDATION FOR
THE DETERMINATION OF HYOSCINE BUTYLBROMIDE AND
OFLOXACIN
HYDROCHLORIDE
IN
BULK
AND
MARKETED
FORMULATIONS”
2
6.0 BRIEF RESUME OF THE INTENDED WORK:
6.1 Need for study:
Analytical Method Development for Pharmaceutical Formulations:
Analytical methods are required to characterize drug substances and drug
products composition during all phases of pharmaceutical development. Development of
methods to achieve the final goal of ensuring the quality of drug substances and drug
products must be implemented in conjunction with an understanding of the chemical
behavior and physicochemical properties of the drug substance. These determinations
require highly sophisticated
instruments
and methods like HPLC,
HPTLC,
Gas
Chromatography and Spectrophotometer etc.
Extensive literature survey reveals that several analytical methods have been
reported for the estimation of hyoscine butylbromide and ofloxacin hydrochloride in
pharmaceutical dosage form which includes Spectrophotometric methods, HPLC and RPHPLC.
Hence there is a need for the development of newer, simple, sensitive, rapid,
accurate and reproducible analytical methods for the routine estimation of hyoscine
butylbromide and ofloxacin hydrochloride in bulk and pharmaceutical dosage form.
6.2 Basic criteria for new method development of drug analysis:

The drug or drug combination may not be official in any pharmacopoeias.

A proper analytical procedure for the drug may not be available in the literature due
to patent regulations.

Analytical methods may not be available for the drug in the form of a formulation
due to the interference caused by the formulation excipients.

Analytical methods for a drug in combination with other drugs may not be
available.

The existing analytical procedures may require expensive reagents and solvents. It
may also involve cumbersome extraction and separation procedures and these may
not be reliable.
3
Analytical method development provides the support to track the quality of the
product from batch to batch. Estimation can be performed by the following two methods:

Titrimetric methods and

Instrumental methods.

Spectrophotometric Methods

Chromatographic Methods
Methods for analyzing drugs in dosage forms can be developed, provided one has
knowledge about the nature of the sample, its molecular weight, polarity, ionic character
and the solubility parameter. Method development involves considerable trial and error
procedures. The most difficult problem usually is where to start, what type of column is
worth trying with what kind of mobile phase and what type of reagent is to be used.

The following is a suggested method development scheme for a typical HPLCUV related substance method.
1. To define the goals for method development (e.g., what is the intended use of the
method?), and to understand the chemistry of the analytes and the drug product.
2. To develop preliminary HPLC conditions to achieve minimally acceptable
separations. These HPLC conditions will be used for all subsequent method
development experiments.
3. To develop a suitable sample preparation scheme for the drug product.
4. To determine the appropriate standardization method and the use of relative
response factors in calculations.
5. To identify the “weaknesses” of the method and optimize the method through
experimental design. Understand the method performance with different conditions,
different instrument set ups and different samples.
6. To complete method validation according to ICH guidelines as mentioned in
Q2 (R1)
4
6.3 DRUG PROFILE OF HYOSCINE BUTYLBROMIDE:1,2,3
Drug category : Antimuscarinic , Anticholinergic agent
Chemical Structure:
IUPAC name:(1S,3s,5R,6R,7S,8r)-6,7-epoxy-8-butyl-3-[(S)-tropoyloxy]tropanium
bromide.
Empirical formula: C21H30BrNO4
Molecular weight: 440.4
Solubility: freely soluble in water and in methylene chloride, sparingly soluble in
anhydrous methanol.
bioavailability: <1%
Protein binding: Low
Half-life: 5 hours
Excretion: Renal (50%) and fecal
Description: a white or almost white, crystalline powder, odourless, or almost odourless.
5
PHARMACOLOGY:3,4
Butylscopolamine,
also
known
as
scopolamine
butylbromide,
butylhyoscine and hyoscine butylbromide, is a peripherally acting antimuscarinic,
anticholinergic agent used as an abdominal-specific antispasmodic. It is a quaternary
ammonium compound and a semisynthetic derivative of scopolamine.
Butylscopolamine is used to treat pain and discomfort caused by abdominal
cramps, menstrual cramps, or other spasmodic activity in the digestive system. It is also
effective at preventing bladder spasms. It is not an analgesic in the normal sense, since it
doesn't 'mask' or 'cover over' the pain, but rather works to prevent painful cramps and
spasms from occurring in the first place. The attachment of the butyl-bromide moiety
effectively prevents the movement of this drug across the blood–brain barrier, effectively
minimizing undesirable CNS side-effects associated with scopolamine/hyoscine.
MECHANISM OF ACTION:
Hyoscine competitively blocks muscarinic receptors and has central and
peripheral actions. It relaxes smooth muscle and reduces gastric and intestinal motility.

SIDE EFFECTS:






Dryness of the mouth
Dyshidrosis (type of skin condition involving small blisters on the hands and feet)
Rash
Itching
Increased heart rate
Producing less sweat than normal etc.
6.4 REVIEW OF LITERATURE:
A literature survey was carried out for the estimation of Hyoscine butylbromide.
It was found that a few methods have been reported for this drug. The collection of
references are reproduced below:

Nouruddin WA, Gamal M, Abdelkawy M5 studied and reported on simultanous
determination of hyoscine butyl bromide and dipyrone in their binary mixture by
RP-TLC spectrodensitometric method. RP-TLC Spectrodensitometric method was
6
developed for determination of Hyoscine Butyl Bromide (HBB) and Dipyrone
(DIP). In this method, HBB and DIP were separated on RP-18 W/ UV254 TLC
plates using developing mobile phase consisting of methanol: citrate buffer
(pH=1.5): triflouroacetic acid (70:30:0.1, by volume) + 0.05 gram of sodium lauryl
sulphate. The obtained bands were then scanned at 210 nm. The proposed method
was successfully applied for determination of HBB and PAR in pure form and in
their pharmaceutical formulations.

Farhadi K and Karimpour A6 studied and reported a new method on
electrochemical behavior and determination of hyoscine-n-butylbromide from
pharmaceutical preparations. The electrooxidation of hyoscine-n-butylbromide
(HBB) was investigated by rotating disk electrode voltammetry, cyclic voltammetry
and controlled potential coulometry in 0.1 M HNO3 and in 0.1 M
tetrabutylammonium perchlorate (TBAP) solutions of acetonitrile at a platinum (Pt)
electrode. Based on the results obtained, it is suggested that a bromide ion of HBB
was oxidized in one reversible step in aqueous solutions and in two reversible steps
in acetonitrile. A differential pulse voltammetric (DPV) method at a Pt electrode
was developed for the determination of HBB in the concentration range of 1.0–10
.6-1.0 – 10.3 M. The procedure was applied to the determination of HBB in its
formulations as well as its recovery from blood serum and urine samples.

Masoud RS, Jokar R7 reported a kinetic spectrophotometric method for trace
amounts determination of bromide in pharmaceutical samples using Janus GreenBromate system. A new simple and rapid kinetic spectrophotometric method has
been developed to trace amounts determination of bromide. This method is based
on the catalytic effect of bromide on the reaction between Janus Green and bromate
in sulfuric acid media. The reaction was followed spectrophotometrically by
measuring the absorbance at 618 nm. The fixed time method was used for the first
210 s. The influence of reagents concentration, temperature and time on the
sensitivity was studied. Under optimum experimental conditions, bromide can be
determined in the range of 10.0-1800.0 μg/L. The relative standard deviations (n =
10) were 0.22 and 0.19% for 100.0 and 1000.0 µg/L of bromide, respectively. The
7
detection limit of the proposed method was 4.1μg/L. The influence of potential
interfering of some ions and biological species on the selectivity was studied. The
proposed method was successfully applied for the determination of bromide in
pharmaceutical samples.

Ojeda CB, Rojas FS8 recent developments in derivative ultraviolet/visible
absorption spectrophotometry derivative spectrophotometry is an analytical
technique of great utility for extracting both qualitative and quantitative information
from spectra composed of unresolved bands, and for eliminating the effect of
baseline shifts and baseline tilts. It consists of calculating and plotting one of the
mathematical derivatives of a spectral curve. Thus, the information content of a
spectrum is presented in a potentially more useful form, offering a convenient
solution to a number of analytical problems, such as resolution of multi-component
systems, removal of sample turbidity, matrix background and enhancement of
spectral details. Derivative spectrophotometry is now a reasonably priced standard
feature of modern micro-computerized UV/Vis spectrophotometry.

Nilgun K, Sumru O, Aysel G9 reported a method on simultaneous determination
of medazepam and hyoscine butylbromide in tablets by second-derivative
ultraviolet spectrometry. A second-derivative UV spectrophotometric method for
the simultaneous determination of medazepam and hyoscine butylbromide in sugarcoated tablets without prior separation is described. In the derivative
spectrophotometric determination of these drugs, calibration graphs were obtained
by plotting peak-trough amplitudes at 252.6 and 264.8 run versus concentration of
medazepam and zero-crossing amplitude at 212.5 nm versus concentration of
hyoscine butylbromide. The relative standard deviation of the method was found to
be ±0.56% for medazepam and ±0.08% for hyoscine butylbromide. This method
has been successfully applied to tablets containing medazepam and hyoscine
butylbromide.

Erk N and Feyyaz O10 reported on Spectrophotometric Simultaneous
determination of analgin and hyoscine n-butyl bromide in sugar-coated tablets two
8
new spectrophotometric methods for the simultaneous determination of analgin and
hyoscine N-butyl bromide in their binary mixture are described. In the first method,
derivative spectrophotometry, the determination of these drugs was performed by
measuring the dA/dλ values at 291.8 nm and 219.8 nm in the first derivative spectra
of the mixture for analgin and hyoscine N-butyl bromide respectively. The relative
standard deviation of the method was found to be 0.08% for analgin and 0.77% for
hyoscine N-butyl bromide. In the second method, the determination of these
compounds in mixture was realized by precipitating hyoscine N-butyl bromide with
ammonium reineckate at pH 6.0 selectively and reading the absorbance of the
solution of the precipitate in acetone at 532.2 nm for hyoscine N-butyl bromide and,
by measuring the dA/dλ values at 306.2 nm in the first derivative spectra of the
remaining solution for analgin. The relative standard deviation of the method was
found to be 0.75% for hyoscine N-butyl bromide and 0.10% analgin. These two
methods have been succesfully applied to a sugar-coated tablet containing hyoscine
N-butyl bromide and analgin.
6.5 DRUG PROFILE OF OFLOXACIN HYDROCHLORIDE:11,12,13
Drug category: Antibacterial agent.
Chemical Structure:
. HCl
IUPAC name : (RS)-9-fluoro-3-methyl-10-(4-methylpiperazin-1-yl)-7-oxo-2,3-dihydro7H-pyrido[1,2,3,-de]-1,4-benzoazeine-6-carboxylic acid hydrochloride
Formula : C18H20FN3O4.HCl
9
Molecular weight : 397.83
Solubility: freely soluble in water and glacial acetic acid.
Bioavailability : 98%
Protein binding : 32%
Half life : 9 hours
Excretion : Renal
Description: A pale yellow or bright yellow crystalline powder.
PHARMACOLOGY:11,14
Ofloxacin is a synthetic chemotherapeutic antibiotic of the fluoroquinolone drug
class considered to be a second-generation fluoroquinolone.
Ofloxacin was first patented in 1982 (European Patent Daiichi) and received
approval from the U.S. Food and Drug Administration (FDA) on December 28, 1990.
Ofloxacin is sold under a wide variety of brand names as well as generic drug equivalents,
for oral and intravenous administration. Ofloxacin is also available for topical use, as eye
drops and ear drops.
Ofloxacin is a racemic mixture, which consists of 50% levofloxacin (the
biologically active component) and 50% of its “mirror image” or enantiomer
dextrofloxacin. When levofloxacin disks were not available in early clinical trials, a 5-pg
ofloxacin disk was substituted. The U.S. Food and Drug Administration (FDA) medical
reviewers considered the two drugs to be one and the same and hence interchangeable.
MECHANISM OF ACTION:
Ofloxacin hydrochloride inhibits the enzyme bacterial DNA gyrase, which nicks
double-stranded DNA, introduces negative supercoils and then reseals the nicked ends.
This is necessary to prevent excessive positive supercoiling of the strands when they
separate to permit replication or transcription. The DNA gyrase consists of two A and two
B subunits: The subunit carries out nicking of DNA, B subunit introduces negative
supercoil and then A subunit reseals the strands.
10
Ofloxacin hydrochloride binds to A subunit with high affinity and interferes with
its strand cutting and resealing function. Recent evidence indicates that in gram positive
bacteria the major target of ofloxacin hydrochloride action is a similar enzyme
topisomerase IV which nicks and seperates daughter DNA strands after DNA replication.
Greater for topisomerase IV may confer high potency against gram positive bacteria. The
bactericidal action probably results from digestion of DNA by exonucleases whose
production is signalled by the damaged DNA.
In place of DNA gyrase or topisomerase IV, the mammalian cells posses an
enzyme topisomerase II that also removes positive supercoils which has very low affinity
for ofloxacin hydrochloride. Hence the low toxicity to host cells.
SIDE EFFECTS:

Hepatotoxicity

Vasculitis

Tendinopathy

Hematologic reactions (including agranulocytosis, thrombocytopenia), and renal
toxicities may occur after multiple doses.
6.6 REVIEW OF LITERATURE:
A literature survey was carried out for the estimation of ofloxacin hydrochloride.
It was found that a few methods have been reported for this drug. The collection of
references are reproduced below:

Rao KS, Banerjee A, Keshar NK15 carried out studies on spectrophotometric
methods for the simultaneous estimation of ofloxacin and tinidazole in bulk and
pharmaceutical dosage form. Their work dealt with the simultaneous estimation of
Ofloxacin (OFL) and Tinidazole (TNZ) in bulk and pharmaceutical dosage form,
without prior separation, by three different techniques (Simultaneous equation,
Absorbance ratio method and First order derivative method). The first method is the
application of simultaneous equation. Where the linearity ranges for OFL and TNZ
were 5-30 μg/ml and 10-50 μg/ml respectively. The second method is the
determination of ratio of absorbance at 278nm, the maximum absorption of TNZ
and isobestic wavelength 283 nm, the linearity ranges for OFL and TNZ were 5-30
11
μg/ml and 10-50μg/ml respectively. The third method is the first order derivative
method, where the linearity ranges for OFL and TNZ were 5-30 μg/ml and 10-50
μg/ml respectively. The results of the analysis have been validated statistically and
by recovery studies, where the percentage recovery was found to be 100.9±0.49 and
97.30±0.20 using the simultaneous equation method, 98±0.45 and 100.4±0.48 using
the graphical absorbance ratio method and 99.10±0.40 and 84.70±0.70 using first
derivative method, for OFL and TNZ respectively.
 Singh R, Maithani M, Saraf SK, Saraf S, Gupta RC16 carried out Simultaneous
Estimation of Ciprofloxacin Hydrochloride, Ofloxacin, Tinidazole and Ornidazole
by Reverse Phase – High Performance Liquid Chromatography accurate isocratic
reverse phase high performance liquid chromatography assay has been developed
for simultaneous estimation of Ciprofloxacin Hydrochloride, Ofloxacin, Tinidazole
and Ornidazole in tablet formulations. The separation was achieved by using C-18
column (RP-18, 5µ) coupled with a guard column of same material, in isocratic
mode with mobile phase mixture of Acetonitrile: Water: Tri ethylamine (25:75:1).
The pH of mobile phase was adjusted to 6.0 ± 0.1 with 50% ortho phosphoric acid.
The flow rate was 1.0 mLmin-1 and the separated drugs were detected using UV
detector at the wavelength of 300 nm. The retention time of Ciprofloxacin
Hydrochloride, Ofloxacin, Tinidazole, and Ornidazole was noted to be 2.7, 3.5, 4.5,
and 5.8 minutes, respectively, indicative of rather shorter analysis time (within 6
minutes). The method was validated with respect to stability, specificity, accuracy,
precision, linearity, range, LOD and LOQ.

Hopkala H, Kowalczuk D17 reported on Application of derivative UV
spectrophotometry for the determination of ciprofloxacin, norfloxacin and ofloxacin
in tablets. The first-, second-, third- and fourth-order derivative spectrophotometric
methods, by using the "peak-zero" (P-0) and "peak-peak" (P-P) techniques of
measurement have been developed for the determination of ciprofloxacin
hydrochloride, norfloxacin and ofloxacin in tablets. The calibration curves were
found to be linear within the concentration range of 2.0-12.0 micrograms ml-1 for
ciprofloxacin hydrochloride, 1.0-10.0 micrograms ml-1 for norfloxacin and 2.5-
12
15.0 micrograms ml-1 for ofloxacin.

Incilay S, Ayla T18 reported the Application of bromophenol blue and bromocresol
purple for the extractive-spectrophotometric determination of ofloxacin Simple,
rapid, and extractive spectrophotometric methods were developed for the
determination of ofloxacin in bulk and pharmaceutical dosage form. These methods
were based on the formation of yellow ion-pair complexes between the basic
nitrogen of the drug and bromophenol blue and bromocresol purple as
sulphonphthalein dyes in phthalate buffer pH 3.0 and pH 3.1, respectively. The
formed complexes were extracted with chloroform and measured at 414 nm for
ofloxacin-bromophenol blue and 408 nm for ofloxacin-bromocresol purple. The
analytical parameters and their effects on the reported systems were investigated.
The reactions were extremely rapid at room temperature and the absorbance values
remains unchanged at 48 hour for ofloxacin-bromophenol blue and 72 hour for
ofloxacin-bromocresol purple. Beer's law was obeyed in the ranges 0.87-17.35 and
0. 58 - 14. 46 μg mL-1 for ofloxacin-bromophenol blue and ofloxacin-bromocresol
purple, respectively. The composition of the ion pairs was found 1:1 by Job's
method. The proposed methods have been applied successfully for the analysis of
the drug bulk form and its dosage form. The results obtained by the proposed
methods were compared and statistical analysis showed no significant difference
between the proposed methods.

Feng Y, Zhao F, Tong S19 studied on the charge transfer reaction of ofloxacin.
This developed spectrophtometric method was based on the charge transfer reaction
for the determination of ofloxacin. The molar absorptivity of the complex at 409
nm was 2.8×104 L·mol -1 ·cm -1. Beer’s law was found to be obeyed in the range
of 0～12 μg/mL -1 of ofloxacin. The relative standard deviation was found to be
0.72%.The composition of the complex was found to be 1∶1 by slope ratio and Bent
French methods.

Tong C, Xiang G, Huang D, Liu W20 reported on determination of ofloxacin
terbium (III) ion fluorescence probe sensitised by surfactant The experiments
13
indicated that terbium (III) ion could complex with the ofloxacin, then emitted the
characteristic fluorescence of terbium (III) ion. While the surfactant of sodium
dodecylbenzene sulfonate (SDBS) was added, the fluorescence intensity of the
system was greatly increased. Based on this, a sensitive method of determining the
ofloxacin was established. The fluorescence intensity was determined by a 1 cm
quartz cell with the excitation wavelengths of 300 nm and the emission
wavelengths of 545 nm. The optimal conditions were obtained as follows:
pH=5.5～6.5, the concentration of terbium was 5.0×10.5mol/L, the surfactant
concentration of SDBS was 5.0×10.4 mol/L. The linear range was 2.0×10.6
～5.0×10.8 mol/L; the detection limit was 6.0×10.9 mol/L.

El-Brashy
AM,
El-Sayed
MM,
and
El-Sepai
FA21
reported
two
spectrophotometric determinations of some fluoroquinolone antibacterials through
charge-transfer and ion-pair complexation reactions. They studied on three
fluoroquinolones namely levofloxacin, norfloxacin and ciprofloxacin have been
performed either in pure form or in their tablets. In the first method, levofloxacin
and norfloxacin are directly treated with bromocresol green (BCG) in
dichloromethane while ciprofloxacin is allowed to react with the same dye in
aqueous acidic buffer. Highly yellow colored complex species were formed
instantaneously in case of levofloxacin and norfloxacin or after extraction into
dichloromethane for ciprofloxacin. The formed complexes were quantified
spectrophotometrically at their absorption maxima at 411 nm for levofloxacin and
412 nm for norfloxacin and ciprofloxacin. The second method involves the reaction
of levofloxacin with chloranilic acid (CA) and norfloxacin with tetracyanoethylene
(TCNE) in acetonitrile to give complexes with maximum absorbance at 521 and
333 nm for the two drugs, respectively. Adopting the first procedure, calibration
graphs were linear over the range 1- 20 µg/mL with mean percentage recoveries of
100.41 ± 0.72, 99.99 ± 0.54 and 100.23 ± 0.91 for the three drugs, respectively. For
the second procedure, the concentration ranges were 15-250µg/mL for levofloxacin
using CA and 0.8-16 µg/mL for norfloxacin using TCNE with mean percentage
recoveries of 99.88 ± 0.45 and 100.26 ± 0.68 for the two drugs, respectively. The
proposed methods were successfully applied to determine these drugs in their tablet
formulations and the results compared favorably to that of reference methods.
14

Salem H, Fada L and Khater W22 studied and reported on spectrofluorimetric
determination of certain fluoroquinolones through charge transfer complex formation.
A highly sensitive spectrofluorimetric method was developed for the analysis of ten
fluoroquinolones (FQs) antibacterials, namely amifloxacin (AMI), ciprofloxacin
(CIP), difloxacin (DIF), enoxacin (ENO), enrofloxacin (ENR), lomefloxacin (LOM),
levofloxacin (LEV), norfloxacin (NOR), ofloxacin (OFL) and pefloxacin (PEF) in
their pharmaceutical dosage forms or in biological fluids through charge transfer
(CT) complex formation with bromanil (BRO). The BRO was found to react with
these drugs to produce stable complexes and the fluorescence intensity of the
complexes was greatly enhanced. The formation of such complexes was also
confirmed by ultraviolet-visible measurements.
The different
experimental
parameters that affect the fluorescence intensity were carefully studied. At the
optimum reaction conditions, the drug-BRO complexes showed excitation maxima
ranging from 275 to 290 nm and emission maxima ranging from 450 to 470 nm.
Rectilinear calibration graphs were obtained in the concentration range 0.02 to 3.1
μg.mL-1 for the studied drugs. The method has been successfully applied to
determine their pharmaceutical dosage forms with good precision and accuracy
compared to official and reported methods as revealed by t and F-tests. They were
also applied for the determination of studied drugs in human urine samples.
6.7 Objective of the Study:
 To develop a new method for estimation of hyoscine butylbromide.
 To develop a new method for estimation of ofloxacin hydrochloride.
 To apply validated method for the estimation of hyoscine butylbromide, and
ofloxacin hydrochloride in pharmaceutical formulation.
 To develop a validated method according to ICH guidelines.
15
7.0
7.1 Materials and Methods:
 Drug: Hyoscimine butylbromide and ofloxacin hydrochloride.
 Reagents:

1,10- phenanthroline

Bromothymol blue

Indigo carmine

Folin ciocaltaeau

3-methyl- 2- benzothiazoline hydrazone(MBTH)
Method development:
 All experiments will be carried out in the Department of Quality Assurance.
Srinivas college of Pharmacy, Mangalore.
 Pure samples of hyoscine butylbromide and ofloxacin hydrochloride will be
procured from Industries involved in bulk manufacture of this drug.
 Dosage formulations will be procured from local market.
 The methods will be developed and validated in Q.A. lab of Srinivas college of
Pharmacy.
 The methods will be developed and validated in Q.A. lab of Srinivas college of
Pharmacy.
 The methods will be first developed, then Validated as per ICH guidelines, then
the method will be applied to the formulations.
 UV spectrophotometer Shimadzu-UV1700 with spectral band width of 2nm and
10nm and matched quartz shall be used for measuring absorbance for Hyoscimine
butylbromide and Ofloxacin hydrochloride solutions.
 UV-Visible spectrophotometer Shimadzu-UV1700 with spectral band width of
2nm and 10nm and matched quartz will be used for measuring absorbance of drug
solutions.
 HPLC instrument JASCO ISOCRATIC HPLC-2000 SYSTEM with C18 column shall
be used.
16
7.2 SOURCES OF DATA:
 References from library – Srinivas College of Pharmacy, Valachil, Mangalore.
 www.pharmainfo.net.
 www.google.com
 www.sciencedirect.com
 www.rxlist.com
 www.pubmed.com
 www.medline.com
 www.wikipedia.com
7.3 Does the study require any investigation to be conducted on patients or animals?
No
7.4 Has the ethical clearance been obtained from your institution in case of 7.3?
Not applicable
17
8.0 REFERENCES:
1) Indian pharmacopoeia 2007; volume 2: 591
2) British pharmacopoeia. 2006; volume 2: 3071
3) http://en.wikipedia.org/wiki/Butylscopolamine
4) Tripathi KD. Essentials of medical pharmacology; Sixth edition 2008: 116
5) Nouruddin WA, Gamal M, Abdelkawy M. Simultanous determination of hyoscine
butylbromide and dipyrone in their binary mixture by RP-TLC spectrodensitometric
method. Int J Chem Anal Sci 2012; 3(10) : 1578-1582
6) Farhadi K and Karimpour A. Electrochemical behavior and determination of
hyoscine-n-butylbromide from pharmaceutical preparations. J Chin Chem Soc
2007; 54: 165-172
7) Masoud RS, Jokar R. Kinetic spectrophotometric method for trace amounts
determination of bromide in pharmaceutical samples using janus green-bromate
system. Int J Ind Chem 2011; 2(1)
8) Ojeda CB, Rojas FS. Recent developments in derivative ultraviolet/visible
absorption spectrophotometry. Anal Chim Acta 2004; 1-24
9) Nilgun K, Sumru O, Aysel G. Simultaneous determination of medazepam and
hyoscine butylbromide in tablets by second-derivative ultraviolet spectrometry. Il
Farmaco 1998; 5(1): 62-64
10) Erk N and Feyyaz O. Spectrophotometric simultaneous determination of analgin and
hyoscine n-butyl bromide in sugar-coated tablets. Analytical letters 1996; 29(3):
369-380
18
11) http://en.wikipedia.org/wiki/ofloxacin
12) http://www.chemicalbook.comofloxacinhydrochloride
13) Indian pharmacopoeia 2007;volume 2: 854
14) Tripathi KD. Essentials of medical pharmacology; Sixth edition 2008: 688
15) Rao KS, Banerjee A, Keshar NK. Spectrophotometric methods for the simultaneous
estimation of ofloxacin and tinidazole in bulk and pharmaceutical dosage form.
Chronicles of young scientist 2011; 2(2): 98-102
16) Singh R, Maithani M, Saraf SK, Saraf S, Gupta RC. simultaneous estimation of
ciprofloxacin hydrochloride, ofloxacin, tinidazole and ornidazole by reverse phase –
high performance liquid chromatography. Eurasian J Anal Chem 2009; volume 4( 2)
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9.0
SIGNATURE OF THE CANDIDATE
10.0 REMARKS OF THE GUIDE
10.1
NAME AND DESIGNATION OF GUIDE
[KERKAR SANAM SAULO]
THE CANDIDATE IS WORKING
UNDER MY DIRECT SUPERVISION IN
LABORATORY OF SRINIVAS
COLLEGE OF PHARMACY,
MANGALORE-574143.
DR. E.V.S. SUBRAHMANYAM,
PROFESOR AND HEAD OF
DEPARTMENT,
DEPARTMENT OF QUALITY
ASSURANCE,
SRINIVAS COLLEGE OF PHARMACY.
10.2 SIGNATURE OF GUIDE
[DR. E.V.S SUBRAHMANYAM]
11.0 HEAD OF THE DEPARTMENT
PROF. Dr. E.V.S SUBRAHMANYAM,
DEPARTMENT OF QUALITY
ASSURANCE,
SRINIVAS COLLEGE OF PHARMACY.
11.1 SIGNATURE OF HOD
[DR. E.V.S SUBRAHMANYAM]
FORWARDED AND RECOMMENDED
12.0
REMARKS OF THE PRINCIPAL
FOR FAVORABLE CONSIDERATION.
DR.RAMAKRISHNA SHABARAYA A.
12.1 SIGNATURE OF THE PRINCIPAL
PRINCIPAL AND DIRECTOR ,
HEAD OF DEPARTMENT,
DEPARTMENT OF PHARMACEUTICS,
SRINIVAS COLLEGE OF PHARMACY,
VALACHIL, MANGALORE.
21