RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES by x8LrHTV

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									   “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        HEERA NAMBIAR
     AND ADDRESS:
                                  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     05 MAY 2011
     COURSE:


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”.




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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 48.6-
       486.0 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 high-
       performance 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 high-
       performance     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, re-
       equilibrate 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]-N-
    ethyl-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).




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        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)




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