“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

1.0

NAME AND ADDRESS OF

THE CANDIDATE

KERKAR SANAM SAULO

I YEAR M. PHARM,

DEPARTMENT OF Q.A.,

SRINIVAS COLLEGE OF PHARMACY,

VALACHIL,POST PARENGIPETE,

MANGALORE -574143

2.0 NAME OF THE

INSTITUTION

SRINIVAS COLLEGE OF PHARMACY,

VALACHIL, MANGALORE.

3.0 COURSE OF STUDY &

SUBJECT

MASTER OF PHARMACY

(QUALITY ASSURANCE)

4.0 DATE OF ADMISSION 26TH MAY 2012

5.0 TITLE OF THE TOPIC:

“ANALYTICAL METHOD DEVELOPMENT AND VALIDATION FOR THE

DETERMINATION OF HYOSCINE BUTYLBROMIDE AND OFLOXACIN

HYDROCHLORIDE IN BULK AND MARKETED FORMULATIONS”

6.0 BRIEF RESUME OF THE INTENDED WORK:

2

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 RP-

HPLC.

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.

Analytical method development provides the support to track the quality of the

3

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 HPLC-

UV 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.

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

5

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

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

6

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 Green-

Bromate 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

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

7

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 sugar-

coated 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

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

8

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-dihydro-7H-pyrido[1,2,3,-de]-1,4-benzoazeine-6-carboxylic acid hydrochloride Formula : C18H20FN3O4.HCl

Molecular weight : 397.83

Solubility: freely soluble in water and glacial acetic acid.

Bioavailability : 98%

Protein binding : 32%

9

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.

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

10

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

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.

11

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-

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

12

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

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

13

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.

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

14

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.

7.1 Materials and Methods:

Drug: Hyoscimine butylbromide and ofloxacin hydrochloride.

Reagents:

1,10- phenanthroline

Bromothymol blue

15

7.0

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.

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

16

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

REFERENCES:

1) Indian pharmacopoeia 2007; volume 2: 591

2) British pharmacopoeia. 2006; volume 2: 3071

3) http://en.wikipedia.org/wiki/Butylscopolamine

17

8.0

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

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

18

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)

17) Hopkala H, Kowalczuk D. Application of derivative UV spectrophotometry for the

determination of ciprofloxacin, norfloxacin and ofloxacin in tablets. Acta Pol Pharm

2000; 57(1):3-13.

18) Incilay S, Ayla T. Application of bromophenol blue and bromocresol purple for the

extractive-spectrophotometric determination of ofloxacin. Analytical letters 2003; 36

(6):1163-1181

19) Feng Y, Zhao F, Tong S. Study on the charge transfer reaction of ofloxacin. J Anal

Sci 2000

20) Tong C, Xiang G, Huang D, Liu W. Determination of ofloxacin by the terbium(Ⅲ)

ion fluorescence probe sensitized by the surfactant. Chin J Anal Chem 2004

21) El-Brashy AM, El-Sayed MM, and El-Sepai FA. Spectrophotometric determination

of some fluoroquinolone antibacterials through charge-transfer and ion-pair

complexation reactions. Bull Korean Chem Soc 2004; Vol. 25 ( 3): 365-372

22) Salem H, Fada L and Khater W. spectrofluorimetric determination of certain

fluoroquinolones through charge transfer complex formation. Am J Pharmacol

Toxicol 2007; Vol. 2 ( 1): 18-25

19

20

9.0 SIGNATURE OF THE CANDIDATE [KERKAR SANAM SAULO]

10.0 REMARKS OF THE GUIDE

THE CANDIDATE IS WORKING UNDER MY DIRECT SUPERVISION IN LABORATORY OF SRINIVAS COLLEGE OF PHARMACY, MANGALORE-574143.

10.1 NAME AND DESIGNATION OF

GUIDE

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]

12.0

REMARKS OF THE PRINCIPAL

FORWARDED AND

RECOMMENDED FOR FAVORABLE

CONSIDERATION.

12.1

SIGNATURE OF THE PRINCIPAL

DR.RAMAKRISHNA SHABARAYA

A.

PRINCIPAL AND DIRECTOR ,

HEAD OF DEPARTMENT,

21

DEPARTMENT OF

PHARMACEUTICS, SRINIVAS

COLLEGE OF PHARMACY,

VALACHIL, MANGALORE.

22