“ DEVELOPMENT OF NEW METHODS FOR THE DETERMINATION OF DARIFENACIN AND OXYBUTYNIN 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
PATEL ROHITBHAI VADILAL
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.
2010 – 2012
1. / NAME OF THE CANDIDATEAND ADDRESS: /
PATEL ROHITBHAI VADILAL
14,RADHAKRISHNA PARK SOCIETY
NEAR GAYATRI MANDIR ROAD,
B/H G.E.B, PATAN-384265 ,GUJRAT
2. / NAME OF THE INSTITUTE: / SRINIVAS COLLEGE OF PHARMACY,
VALACHIL, FARANGIPETE (POST),
MANGALORE-574143.
3. / COURSE OF THE STUDY AND SUBJECT: /
MASTER OF PHARMACY
(QUALITY ASSURANCE)
4. / DATE OF ADMISSION TO THE COURSE: / 29th Oct. 20105. / TITLE OF THE TOPIC:
“DEVELOPMENT OF NEW METHODS FOR THE DETERMINATION OF DARIFENACIN AND OXYBUTYNIN IN BULK AND MARKETED FORMULATIONS AND THEIR VALIDATION”.
RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES
BANGALORE, KARNATAKA
ANNEXURE – II
PROFORMA FOR REGISTRATION OF SUBJECTS FOR DISSERTATION
6. / BRIEF RESUME OF THE INTENDED WORK:6.1 Need for the Study: 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 Darifenacin and Oxybutynin. Our intention is to develope a economic, less time consuming method for the estimation of Darifenacin and Oxybutynin 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 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 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)
6.3 General discussion on Darifenacin: 1a
Drug category : Selective M3 muscarinic acetylcholine receptor antagonist
Chemical Structure:
Ø IUPAC Name: (S)-2-[1-[2-(2, 3-dihydrobenzofuran-5-yl)ethyl] pyrrolidin-3-yl] -2,2-diphenyl-acetamide.
Ø Empirical formula: C28H30N2O2
Ø Physical state: White or almost white powder
Ø Molecular mass: 426.55 g/mol
Ø Solubility: Freely soluble in methanol & insoluble in n-hexane, soluble in water.
Darifenacin is a generic medication commonly marketed under the brand name Enablex®(7.5 mg and 15 mg Derifenacin). It is prescribed to treat urinary incontinence or an overactive bladder. This drug works to reduce muscle spasms that may occur in the urinary tract and bladder, which can decrease symptoms like frequent and urgent urination, as well as uncontrollable urination. Darifenacin is classified as an antimuscarinic.1b
Mechanism of action:1a Darifenacin works by blocking the M3 muscarinic acetylcholine receptor, which is primarily responsible for bladder muscle contractions. It thereby decreases the urgency to urinate. It should not be used in people with urinary retention.
6.4 Review of Literature:
A literature survey was carried out for the estimation of Darifenacin. It was found that a few methods have been reported for this drugs. The collection of references are reproduced below;
· RadhakrishnanandP et al;2 A Validated LC Method for determination of the Enantiomeric purity of Darifenacin in bulk drug and Extended Release Tablets. Normal phase chromatographic separation was performed on an immobilized cellulose based chiral stationary phase (Chiralpak-IC) with n-hexane:ethanol:diethylamine (50:50:0.3, v/v/v) as mobile phase at a flow rate of 1.0 mL min-1. The elution time was ∼15 min. The resolution (Rs) between the enantiomers was greater than four and interestingly the (R)-enantiomer was eluted prior to Darifenacin in the developed method. The limit of detection (LOD) and limit of quantification (LOQ) for the (R)-enantiomer were 0.02 μg and 0.07 μg, respectively, for a 10 μL injection volume.
· KAYEB. et al 3 Rapid, solid phase extraction technique for the high-throughput assay of darifenacin in human plasma. The newly designed, 96-tube micropreparation block facilitates high throughput by enabling the extraction of 96 samples simultaneously. The system is described, linked to HPLC/APCI-MS/MS, for the determination of Darifenacin in human plasma. The resulting procedure, using deuterated Darifenacin as internal standard, is validated over the concentration range 25-2000 pg/mL ; accuracy (0.6-4.6%) and precision (3.6-18.8%) are considered acceptable and overall recovery was determined to be ∼50%.
· Pravin PS, Jagathi V, Saibabu S4 have developed a visible spectrophotometry method for Darifenacin by using bromo thymol blue (λ max-415 nm) and picric acid(λ max-400 nm) the absorbance of chromogen is measured against the correspondig reagent blank.
· NK Sathish, Pradip V, IA Chethan 5 have developed a uv spectrophotometry method for estimation of Darifenacin hydrobromide. The method is based on the Darifenacin hydrobromide showing absorbance at 286 nm in methanol. This method is obey beer’s law in the concentration range of 10-100 µg/ml. The % recovery is grater than 98%.
· Pravin PS, Devala Rao G, Jaghathi V, Shaiba M6 have developed a spectrofluorimetry method for estimation of Darifenacin in pure and pharmaceutical formulation. Beer’s law is abeyed in the concentration range of 1-5 µg/ml in methanol in excitation wave leangth at 292 nm and emission wave leangth at 314 nm with good correlation co-efficient 0.9995.
6.5 General discussion on Oxybutynin:7a
Drug category: Antimascarinic antagonist
Chemical Structure:
IUPAC Name: 4-Diethylaminobut- 2-ynyl2- cyclohexyl-2- hydroxy-2-phenyl-ethanoate
Empirical formula: C22H31NO3
Physical state: White to off-white crystaline powder
Molecular mass: 357.486 gm/mol
Solubility: soluble in water, freely soluble in methanol, soluble in acid but not soluble in alkali
Oxybutynin is a belongs to class of antimuscarinic antagonist. Oxybutynin chloride relaxes bladder smooth muscle. In patients with conditions characterized by involuntary bladder contractions, cystometric studies have demonstrated that Oxybutynin chloride increases bladder (vesical) capacity, diminishes the frequency of uninhibited contractions of the detrusor muscle, and delays the initial desire to void. Oxybutynin chloride thus decreases urgency and the frequency of both incontinent episodes and voluntary urination7b.
Mechanism of action: 7c Oxybutynin has a dual mechanism of action. Contraction of the smooth muscle of the bladder is stimulated by the release of acetylcholine by the nerves within the bladder and the attachment of the acetylcholine to receptors on the surface of the muscle cells. Oxybutynin suppresses involuntary contractions of the bladder's smooth muscle (spasms) by blocking the release of acetylcholine. This is called an "anticholinergic effect." Oxybutynin also directly relaxes the bladder's outer layer of muscle (the detrusor muscle). 6.6 Review of Literature:
A literature survey was carried out for estimation methods of Oxybutynin. It was found that a very few methods have been reported for this drug. The collection of references are reproduced below:
· Shrikanth K, Emmanuel KA, Raju R8 have developed a spectrophotometry method for determination of Oxybutynin through ion association complex formation. Method involves complex formation of Oxybutynin chloride with tropaeoline OOO(TPOOO) and Alizarin Red-S(ARS). It has absorption max. at 480 nm and 420 nm respectively and obeyed beer’s law in the concentration range 1.0-7.5 µg/ml and 2.0-15 µg/ml respectively.both have correlation co-efficient value of 0.9999.
· Varma VS, Kaushal AM, Garg S9 have developed a A new UV spectrophotometric method and a reversed-phase HPLC method were developed for quantitative evaluation of Oxybutynin hydrochloride (OXB) formulations. Determination of OXB by UV spectroscopic method was based on complexation of OXB with picric acid to form picrate, which was extracted to chloroform. The picrate complex showed quantifiable absorbance at 344nm. Chromatography was carried out at 25 degrees C on a 4.6mm x 250mm 5microm cyano column that contained USP packing L10 with water:methanol:acetonitrile::48:12:40 (v/v), as mobile phase. UV detector was set at 203nm. The LOD and LOQ of HPLC method were 0.5 and 1.65microg/ml, respectively.
· Wagieh NE, Hegazy MA, Abdelkawy M, Abdelaleem EA10 have developed a simple, accurate, sensitive and validated UV spectrophotometric, chemometric and HPTLC-densitometric methods were developed for determination of Oxybutynin hydrochloridein presence of its degradation product and additives in its pharmaceutical formulations. Method A is the first derivative of ratio spectra (DD(1)) which allows the determination of Oxybutynin in presence of its degradated in pure form and tablets by measuring the peaks amplitude at 216nm. Method B and C are principal component regression (PCR) and partial least-squares (PLS) for determination of Oxybutynin in presence of its depredated in pure form, tablets and syrup. While, the developed high performance thin layer chromatography HPTLC-densitometric method was based on the separation of Oxybutynin from its degradation product, methylparaben and propylparaben followed by densitometric measurement at 220 nm which allows the determination of Oxybutynin in pure form, tablets and syrup. The separation was achieved using HPTLC silica gel F(254) plates and chloroform:methanol:ammonia solution:triethylamine (100:3:0.5:0.2, v/v/v/v) as the developing system.
6.7 Objective of the Study:
Ø Darifenacin and Oxybutynin is not a official in pharmacopoeias like IP, BP and USP hence no official method is available for the estimation of this drug.
Ø To develope a new method for estimation of Darifenacin.
Ø To develope a new method for estimation of Oxybutynin.
Ø To develope a validated method according o ICH guidelines.
Ø To apply validated method for the estimation of Darifenacin, and Oxybutynin in pharmaceutical formulation.
7. / 7.1 Materials and Methods:
Ø Drug: Darifenacin and Oxybutynin.
Ø Reagents: Methanol, acetonitrile, potassium permanganate, potassium dichromate, acetic acid. hydrochloric acid.
Method development:
Ø All experiments will be carried out in the Department of Quality Assurance. Srinivas college of Pharmacy, Mangalore.
Ø Pure samples of Darfenacin and Oxybutynin, will be procured from Industries involved in bulk manufacture of this drug.
Ø Dosage formulations will be procured from local market.
Ø 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.
Ø HPLC instrument JASCO ISOCRATIC HPLC-2000 SYSTEM with C18 column shall be used.
7.2 Source of Data:
Ø References from library –srinivas college of pharmacy
Ø www.pharmainfo.net
Ø www.google.com
Ø 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--
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://pubchem.ncbi.nlm.nih.gov/summary/summary.cgi?cid=444031 (b)http://www.wisegeek.com/what-is-darifenacin.htm
2) RadhakrishnanandP et al; A Validated LC Method for Determination of the Enantiomeric Purity of Darifenacin in Bulk Drug and Extended Release Tablets. J of Chromatographia ISSN0009-5893 CODENCHRGB7; 2008;68(11-12):1059-62
3) KAYEB. et al; Rapid, solid phase extraction technique for the high-throughput assay of Darifenacin in human plasma. J of Analytical chemistry ISSN0003-2700 CODENANCHAM; 1996;68(9):1658-60
4) Pravin PS, Jagathi V, Saibabu S ; Visible spectrophotometry method for the estimation of Darifenacin. Research J of pharmaceutical, biological and chemical sciences ISSN 0975-8585 CODEN RJBPCS; Apr-2010,1(2):254
5) Sathish NK, Pradip V, Chethan IA ; UV spectrophotometry method for estimation of Darifenacin hydrobromide in marketed formmulation. Palagia research library, Der pharmacia sinica ISSN O976-8688, CODEN(USA) PSHIBD; 2011, 2(2):169-76
6) Pravin PS, Devala Rao G, Jaghathi V, Shaiba M ; Spectrofluorimetry method for estimation of Darifenacin in pure and pharmaceutical formulation. Asian J of pharmaceutical and clinical research, ISSN 0974-2441, 2011,4(1):44-46
7) (a)en.wikipedia.org/wiki/Oxybutynin
(b) http://www.drugs.com/pro/oxybutynin.html
(c)medicinenet,com/oxybutynin
8) Shrikanth K, Emmanuel KA, Raju R ; A spectrophotometry method for determination of Oxybutynin through ion association complex formation. Rasayan J Chem. ISSN 0974-1496, CODEN RJCABP, 2010,3(1):179-87
9) Varma VS, Kaushal AM, Garg S ; A new UV spectrophotometric method and a reversed-phase HPLC method developed for quantitative evaluation of Oxybutynin hydrochloride (OXB) formulations. J of pharmaceutical and biological analysis2004,36(3):669-74
10) Wagieh NE, Hegazy MA, Abdelkawy M, Abdelaleem EA ; UV spectrophotometric, chemometric and HPTLC-densitometric methods were developed for determination of Oxybutynin hydrochloridein presence of its degradation product and additives in its pharmaceutical formulations. Pak J Pharm Sci. Oct 2008,21(4):366-9.
9. / Signature of the Candidate: / (Patel Rohitbhai V.) (((
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,
Profesor 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.
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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