Download annexure – ii - Rajiv Gandhi University of Health Sciences

“DEVELOPMENT OF NEW ANALYTICAL METHODS FOR THE
DETERMINATION OF BROMFENAC AND BIMATOPROST IN BULK AND
MARKETED FORMULATIONS AND THEIR VALIDATION”
MASTER OF PHARMACY
DISSERTATION PROTOCOL
SUBMITTED TO THE
RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES KARNATAKA,
BANGALORE.
BY
HEERA NAMBIAR
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.
2011– 2013
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RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES
BANGALORE, KARNATAKA
ANNEXURE – II
PROFORMA FOR REGISTRATION OF SUBJECTS FOR DISSERTATION
1.
NAME OF THE CANDIDATE
AND ADDRESS:
HEERA NAMBIAR
1st YEAR M.PHARM, DEPT. OF
QUALITY ASSURANCE,
SRINIVAS COLLEGE OF PHARMACY,
VALACHIL, MANGALORE-574143.
2.
NAME OF THE INSTITUTE:
SRINIVAS COLLEGE OF PHARMACY,
VALACHIL, FARANGIPETE (POST),
MANGALORE-574143.
3.
COURSE OF THE STUDY AND
MASTER OF PHARMACY
SUBJECT:
(QUALITY ASSURANCE)
4.
DATE OF ADMISSION TO THE
COURSE:
05 MAY 2011
5.
TITLE OF THE TOPIC:
“DEVELOPMENT
OF
NEW
ANALYTICAL
METHODS
FOR
THE
DETERMINATION OF BROMFENAC AND BIMATOPROST IN BULK AND
MARKETED FORMULATIONS AND THEIR VALIDATION”.
2
6.
BRIEF RESUME OF THE INTENDED WORK:
6.1 Need for the Study1: The number of drugs, which may be either new entities or partial
structural modification of the existing ones, introduced into the market is increasing every
year. Very often there is a time lag from the date of introduction of a drug into the market to
the date of its inclusion in pharmacopoeias. Hence, standards and analytical procedures for
these drugs may not be available in the pharmacopoeias. There is not much data is available
on analytical methods by spectrophotometric, spectrofluorimetric and HPLC methods for
Bromfenac and Bimatoprost. Our intention is to develop an economic, less time consuming
method for the estimation of Bromfenac and Bimatoprost by different analytical methods
such as spectrophotometric, spectrofluorimetric and HPLC.
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
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
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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 use.

The following is a suggested method development scheme for HPLC and UV
methods.
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 drawback 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)
6.3
General discussion on Bromfenac:1
Drug category : Non-steroidal anti-inflammatory drug(NSAID) for ophthalmic use.
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Chemical Structure:
 IUPAC Name: 2-[2-amino-3-(4-bromobenzoyl)phenyl]acetic acid
 Empirical formula: C15H12BrNO3
 Physical state: Yellow powder
 Molecular mass: 334.16 g/mol
 Solubility: Soluble in water, methanol and dilute base.
Bromfenac is a non-steroidal anti-inflammatory drug (NSAID) marketed
in the US as an ophthalmic solution (current brand name Bromday, prior formulation brand
name Xibrom, which has since been discontinued.) by ISTA Pharmaceuticals for short-term,
local use. Bromday is the once-daily formulation of Bromfenac, while Xibrom, which has
since been discontinued, was the twice-daily formulation. Bromfenac is indicated for the
treatment of ocular inflammation and pain after cataract surgery, though it may be prescribed
in an off-label manner by the physician.
Mechanism of action: The mechanism of action of Bromfenac is thought to be due to
its ability to block prostaglandin synthesis by inhibiting cyclooxygenase 1 and 2 (COX-1 and
-2), also called prostaglandin G/H synthase 1 and 2. COX-1 and -2 catalyze the conversion of
arachidonic acid to prostaglandin G2 and prostaglandin G2 to prostglandin H2. Prostaglandin
H2 is the precursor to a number of prostaglandins (e.g. PGE2) involved in fever, pain,
swelling, inflammation, and platelet aggregation. Bromfenac antagonizes COX by binding to
the upper portion of the active site, preventing its substrate, arachidonic acid, from entering
the active site. Prostaglandins have been shown in many animal models to be mediators of
certain kinds of intraocular inflammation. In studies performed in animal eyes,
prostaglandins have been shown to produce disruption of the blood-aqueous humor barrier,
vasodilation, increased vascular permeability, leukocytosis, and increased intraocular
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pressure. The analgesic and anti-inflammatory effects of Bromfenac occurs as a result of
decreased prostaglandin synthesis.
6.4 Review of Literature:
A literature survey was carried out for the estimation of Bromfenac. It was found
that a few methods have been reported for this drug. The collection of references are
reproduced below;

Jia-yi K, Biao WU, Hong-xiao CUI2 have developed RP-HPLC method for
determination of the content of Bromfenac sodium and its related substances.
Method:A C18 column(150 mm×4.6 mm,5 μm) was used with the mobile phase of
0.025 mol·L-1NaH2PO4buffer solution(0.1% triethylamine,adjust pH to 4.0 with
phosphoric acid)-acetonitrile(60∶40) at a flow rate of 1.0 mL·min-1 and the detection
wavelength of 270 nm. Results: The Bromfenac sodium and the related substances
can be completely separated. The calibration curve was linear in the range of 48.6486.0 μg·mL-1with r=0.9997.The detection limit was 0.5 ng. The precision(RSD)was
0.83%.Conclusion:The method for determination of Bromfenac sodium and its
related substances by RP-HPLC is easy to operate and accurate.

Schmitz G, Lepper H, Estler CJ3 have developed a rapid and sensitive highperformance liquid chromatographic method is presented for the determination of
diclofenac and its hydroxylated and methoxylated metabolites. The procedure
describes extraction of diclofenac and its metabolites from acidified incubation
medium into tert.-butylmethyl ether. Separation is achieved with a C18 reversed-phase
column and quantification by UV detection at 280 nm. The method employs an
internal standard resulting in good accuracy and precision. The limit of detection is 5
ng/ml for diclofenac and 10 ng/ml for its metabolites. One analysis requires no more
than twelve minutes so that the assay is very suitable for the determination of a large
number of samples.

Gumbhir-Shah K, Cevallos WH, DeCleene SA, Halstenson CE, Korth-Bradley JM4
have developed method to estimate absolute bioavailability of Bromfenac and to
compare its pharmacokinetics after intravenous and oral administration. This was a
randomized, open-label, single-dose, crossover study conducted under fasting
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conditions with a washout period of at least 48 hours between doses. Each subject
received a 50-mg dose of Bromfenac both intravenously and orally followed by
collection of blood samples at specified time intervals. Bromfenac plasma
concentrations were measured by using a validated HPLC method with ultraviolet
detection.

Boni JP, Cevallos WH, DeCleene S, Korth-Bradley JM5 have developed method To
assess the effect of Bromfenac sodium, a non-narcotic analgesic drug under
development, on the pharmacokinetics and pharmacodynamics of glyburide in
patients with type II diabetes. Bromfenac concentrations were measured by highperformance
liquid
chromatography
with
ultraviolet
detection.
Glyburide
concentrations were measured by gas chromatography with nitrogen-phosphorus
detection. Glycemia was measured repeatedly on day 3 of each treatment.
Pharmacokinetic analysis was performed with non-compartmental techniques. No
significant
differences
in
the
pharmacokinetics
of
glyburide
or
in
the
pharmacodynamic response of serum glucose levels were observed between placebo
and Bromfenac. Intersubject variability of concentrations was modest for glyburide
and glucose, with a CV of 43% or less.

Osman M, Chandrasekaran A, Chan K, Scatina J, Ermer J, Cevallos W, et al6 have
developed HPLC method of Bromfenac. The column used was150 × 4.6 5 μm
HiChrom (Regis) (for electrospray negative ion MS (Finnigan-MAT TSQ 700)
detection use 250 ×2 C18 DB (Supelco) column with the gradient below at 0.2
mL/min)and the mobile phase was Gradient. MeCN:100 mM pH 4.9 ammonium
acetate buffer from 10:90to 45:55 over 50 min, maintain at 45:55 for 10 min, reequilibrate at initial conditions for 15 minutes.
6.5 General discussion on Bimatoprost11 :
Drug category: Antiglaucoma agent (ophthalmic); antihypertensive
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Chemical Structure:
IUPAC Name: 7-[3,5-dihydroxy-2- (3-hydroxy-5-phenyl-pent-1-enyl)- cyclopentyl]-Nethyl-hept-5-enamide
Empirical formula: C25H37NO4
Physical state: Crystalline solid.
Molecular mass: 415.566 gm/mol
Solubility: Very soluble in ethanol and methanol, and slightly soluble in water.
Bimatoprost is a prostaglandin analog/prodrug used topically (as eye drops) to control
the progression of glaucoma and in the management of ocular hypertension. It reduces
intraocular pressure (IOP) by increasing the outflow of aqueous fluid from the eyes. It has
also been used and prescribed off-label to lengthen eyelashes. In December 2008, this use
was approved by the U.S. Food and Drug Administration; Recently, at least three case series
have suggested that Bimatoprost has the ability to reduce adipose (fat) tissue.
Mechanism of action: Antiglaucoma agent—Bimatoprost, a prostamide or synthetic
prostaglandin analog, is thought to lower intraocular pressure (IOP) by increasing the
outflow of aqueous humor through both the trabecular meshwork and uveoscleral drainage
systems.
6.6 Review of Literature:
A literature survey was carried out for estimation methods of Bimatoprost. It was
found that a very few methods have been reported for this drug. The collection of references
are reproduced below:

Murali KP, Thirupathi TR, Bujagendra RM , Narasimha CR, Kishore KR,
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Venkateswarlu P7 have developed A Novel method and determination of a Novel
impurity (methyl ester) in Bimatoprost by LC-MS and chromatographic separation by
Ultra Performance Liquid Chromatography (UPLC). It is used to treat drug for in the
treatment of a topical eye medication used to reduce pressure inside the eye and also
including glaucoma and ocular hypertension, in which increased pressure can lead to
a gradual loss of vision. The separation of Bimatoprost and its isomers 15(R) –
Bimatoprost (impurity I), Bimatoprost acid (impurity II), keto (impurity III) and
Bimatoprost methyl ester (impurity IV) for the first time by UPLC on Aquity UPLC
BEH Shield RP 18 (100 X 2.1 mm), 1.7μm and detector of UV at 237nm, column
with a homogeneous mixture of n-Hexane, dehydrated alcohol and methanol
(80:10:10). The proposed method was found to be specificity, linearity, and precision,
intermediate precision, and accuracy, stability in analytical solution and robustness.
The validation was performed according to the current requirements as laid down in
the ICH guidelines.

Woodward DF, Krauss AHP, Chen J, Liang Y, Li C, Protzman CE, et al8 have
developed HPLC method for Bimatoprost. The column used was 150 × 4.6 5 μm
Ultrasphere IP and mobile phase used was Gradient. A:B 100:0 for 1 min, to 40:60
over 16 min (+ curved), maintain at 40:60 for 4 min, return to initial conditions over 1
min, re-equilibrate at initial conditions for 8 min. A was MeCN:20 mM pH 2.8
potassium phosphate buffer 20:80. B was MeCN:20 mM pH 2.8 potassium phosphate
buffer 50:50.

Suresh SK, Natraj K, Asadulla K, Kalyan BK, Venkateshwara JR9 have developed
RP-HPLC method for the estimation of Bimatoprost in bulk and in ophthalmic
solution. The mobile phase used was a mixture of phosphate buffer (PH 2.8 is
adjusted with orthophosphoric acid) and acetonitrile (55:45 v/v). The wavelength
used for detection of Bimatoprost was 210 nm and flow rate 1 ml/min. Linearity was
determined for Bimatoprost in the range of 50-250 µg/ml. The correlation coefficient
(‘r’) value was found to be 0.9998. The method was validated with respect to
accuracy, precision, linearity and robustness as per the ICH Guidelines. The proposed
method can be successfully used to determine the drug content in marketed
formulation.

Maxey KM, Johnson JL, LaBrecque J10 have developed HPLC and mass
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spectrometry
for
conversion
of
Bimatoprost
to
17-phenyl-18,19,20-trinor
prostaglandin F(2alpha) continued for at least 24 hours after excision of the cornea,
with a conversion rate of approximately 25 microg/24 hours. This hydrolysis product
is identical to the free acid of latanoprost with the exception of a double, rather than a
single, bond at the carbon 13-14 position. Assuming that this conversion also occurs
in vivo at a similar rate, this hydrolysis product may account for the reduction of
intraocular pressure occurring in patients treated with Bimatoprost.
6.7
Objective of the Study:
 Bromfenac and Bimatoprost is not a official in pharmacopoeias like IP, BP and USP
hence no official method is available for the estimation of this drug.
 To develope a new method for estimation of Bromfenac.
 To develop a new method for estimation of Bimatoprost.
 To develop a validated method according o ICH guidelines.
 To apply validated method for the estimation of Bromfenac and Bimatoprost in
pharmaceutical formulation.
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7.
7.1 Materials and Methods:
 Drug: Bromfenac and Bimatoprost.
 Reagents: Methanol, acetonitrile, potassium permanganate, potassium dichromate,
acetic acid. hydrochloric acid.
Method development:
 All experiments will be carried out in the Department of Quality Assurance. Srinivas
college of Pharmacy, Mangalore.
 Pure samples of Bromfenac and Bimatoprost will be procured from Industries
involved in bulk manufacture of this drug.
 Dosage formulations will be procured from local market.
 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.
 Colorimeter instrument Systronics Spectrophotometer 166 shall be used.
7.2 Source of Data:
 References from library –Srinivas College of Pharmacy
 www.pharmainfo.net
 www.google.com
 www.sciencedirect.com
7.3 Does the study require any investigations or interventions to be conducted
On patients or other human or animals? If so please describe briefly:
-- Not applicable--
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7.4 Has the Ethical Clearance been obtained from your Institution in case of 7.3?
-- Not applicable—
8.
LIST OF REFERENCES:
1) a) http://www.wisegeek.com/what-is-bromfenac.htm
b) http://www.pharma info.net
2) Jia-yi K, Biao WU, Hong-xiao CUI. RP-HPLC determination of content and related
substances of Bromfenac sodium. Chinese Journal of Pharmaceutical Analysis 2009;
04-5.
3) Schmitz G, Lepper H, Estler CJ. High-performance liquid chromatographic method
for the routine determination of diclofenac and its hydroxy and methoxy metabolites
from in vitro systems. Journal of Chromatography B: Biomedical Sciences and
Applications 1993; 620(1)22:158-63.
4) Gumbhir-Shah K, Cevallos WH, DeCleene SA, Halstenson CE, Korth-Bradley JM.
Absolute bioavailability of Bromfenac in humans; Ann Pharmacother 1997;
31(4):395-9.
5) Boni JP, Cevallos WH, DeCleene S, Korth-Bradley JM. The influence of Bromfenac
on the pharmacokinetics and pharmacodynamic responses to glyburide in diabetic
subjects. Pharmacotherapy 1997; 17(4):783-90.
6) Osman M, Chandrasekaran A, Chan K, Scatina J, Ermer J, Cevallos W, et al. HPLC
method of Bromfenac. J.Clin.Pharmacol 1998; 38:744–52.
7) Murali KP, Thirupathi TR, Bujagendra RM , Narasimha CR, Kishore KR,
Venkateswarlu P. Determination of a novel impurity by LC-MASS and
chromatographic separation of Bimatoprost, isomers and their impurities by UPLC.
Journal of Pharmacy Research 2011; 4(7):2381-3.
8) Woodward DF, Krauss AHP, Chen J, Liang Y, Li C, Protzman CE, et al.
Pharmacological characterization of a novel antiglaucoma agent, Bimatoprost (AGN
192024), J.Pharmacol.Exp.Ther 2003; 305:772–85.
9) Suresh SK, Natraj K, Asadulla K, Kalyan BK, Venkateshwara JR. Development and
Validation of RP-HPLC Method for Estimation of Bimatoprost in Pharmaceutical
Dosage Forms. Journal of Pharmacy Research 2009; 4(10).
12
10) Maxey KM, Johnson JL, LaBrecque J. The hydrolysis of Bimatoprost in corneal
tissue generates a potent prostanoid FP receptor agonist. Surv Opthalmol
2002;47(1):34-40.
11) http://www.drugs.com/pro/bimatoprost.html
9.
Signature of the Candidate:
(Heera Nambiar) (((
10. Remarks of the Guide:
The candidate is working under my direct supervision in laboratories of Srinivas College of
Pharmacy, Mangalore-574143.
11. 11.1 Name & Designation of the Guide :
Dr. E.V.S Subrahmanyam,
Professor and head,
Department of Quality Assurance,
Srinivas College of Pharmacy.
11.2 Signature of Guide:
(Dr. E.V.S Subrahmanyam)HINI R. M.)
11.3 Head of the Department:
PROF. Dr. E.V.S SUBRAHMANYAM,
DEPARTMENT OF QUALITY ASSURANCE,
SRINIVAS COLLEGE OF PHARMACY.
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11.4 Signature of HOD:
(Dr. E.V.S Subrahmanyam)
12. 12.1 Remark of the Principal:
FORWARDED
AND
RECOMMENDED
FOR
FAVORABLE CONSIDERATION.
12.2 Signature of the Principal
(Dr. A.R SHABARAYA)
14