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The Pharma Review THE UMBRELLA JOURNAL OF THE PHARMA INDUSTRY Section 2 - Analytical Method Development ® Validated Method Development for Estimation of Atorvastatin Calcium and Fenofibrate in Fixed Dose 3 Combination by HPTLC - P. B. Deshpande, G. Shridharan, Libi Anandi, D. Jadhav, M. C. Damle, S. V. Gandhi Simultaneous High Performance Liquid Chromatographic Estimation of AmphotericinB in Dry Injection Dosage Form 6 - J.P. Mall, P.C. Patel And Dr. J.N. Verma Stress Degradation Studies on Ceftazidime Sodium and Development of a Validated Stability-Indicating HPLC Assay 9 - Lalitha N, S.B.Puranik, Sanjay Pai P.N, Rao G.K. Spectrophotometric Methods for Determination of Olmesartan Medoxomil and Hydrochlorothiazide in Tablet Dosage Form 12 - S. S. Kadukara, P. N. Ranjanea, S. S. Ranhera, S. V. Gandhia High Performance Thin Layer Chromatographic Method for Simultaneous Estimation of Amlodipine Besilate and Bisoprolol Fumarate in Pharmaceutical Preparations - R.B. Kakde, V.H. Kotak and D.L.Kale 14 Development And Validation of Spectrophotometric Method for Determination of Montelukast Sodium 18 in Bulk and Tablet Formulation - Varun Pawar, Lalitha N, Puranik SB, Sanjay Pai P.N, Rao G.K. Gas Chromatographic Determination of Residual Solvents in Pharmaceutical Excipients 21 - S. B. Puranik, Varun R. Pawar, N. Lalitha, P. N. Sanjay Pai, G. K. Rao UV-Spectrophotometry and First Order Derivative Methods for Estimation of Efavirenz in Bulk and Capsules 24 - Charushila H. Bhirud, Atul A. Shirkhedkar, Ravindra A. Fursule and Sanjay J. Surana RP-HPLC Estimation of Eplerenone in Tablets - K. P. Bhusari, P. B. Khedekar, N. D. Amnerkar, S. M. Dhole, V. S. Banode 26 Development and Validation of New RP-HPLC Method with UV-Detection for the Determination of Carvedilol 28 in Human Serum - Mogallapalli L V Setti, Vijaya Ratna J Gas Chromatographic Determination of Methanol and Isopropyl Alcohol Impurities in Herbal Extracts 32 - S. B. Puranik, Varun R. Pawar, Lalitha N, P. N. Sanjay Pai, G. K.Rao Simultaneous Spectrophotometric Estimation of Paracetamol and Tramadol Hydrochloride in Solid Dosage Form 36 - P.G. Yeole, S.J. Wadher, M.P. Puranik, R.O. Ganjiwale Atul A. Shirkhedkar, Prasad M. Bugdane, Sanjay J. Surana First Order Derivative Spectrophotometric Determination of Nebivolol in Bulk and Tablets 39 - Contents Bagbania, Baddi-Nalagarh Road, Distt. Solan (HP) - 174 101 E-mail : [email protected], Website : www.unitedbiotechindia.com UNITED BIOTECH (P) LIMITED DIVISIONS AT A GLANCE COPAS ONCOLOGY RINON Division Division Division CRITICAL CARE SPECIALTY HYGEA Division Division Division LIFE is PRECIOUS for the ESSENCE of life WE CARE LIFE is PRECIOUS for the ESSENCE of life WE CARE

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UNITED BIOTECH (P) LIMITEDBagbania, Baddi-Nalagarh Road, Distt. Solan (HP) - 174 101 E-mail : [email protected], Website : www.unitedbiotechindia.com

DIVISIONS AT A GLANCE CRITICAL CARE Division COPAS Division

SPECIALTY Division HYGEA Division

ONCOLOGY Division RINON Division

LIFE is PRECIOUS

WE CARE for the ESSENCE of life

The Pharma ReviewSection 2 - Analytical Method DevelopmentContents Validated Method Development for Estimation of Atorvastatin Calcium and Fenofibrate in Fixed Dose Combination by HPTLC - P. B. Deshpande, G. Shridharan, Libi Anandi, D. Jadhav, M. C. Damle, S. V. Gandhi

THE UMBRELLA JOURNAL OF THE PHARMA INDUSTRY

3 6 9

Simultaneous High Performance Liquid Chromatographic Estimation of AmphotericinB in Dry Injection Dosage Form - J.P. Mall, P.C. Patel And Dr. J.N. Verma Stress Degradation Studies on Ceftazidime Sodium and Development of a Validated Stability-Indicating HPLC Assay - Lalitha N, S.B.Puranik, Sanjay Pai P.N, Rao G.K.

Spectrophotometric Methods for Determination of Olmesartan Medoxomil and Hydrochlorothiazide in Tablet Dosage Form 12 - S. S. Kadukara, P. N. Ranjanea, S. S. Ranhera, S. V. Gandhia High Performance Thin Layer Chromatographic Method for Simultaneous Estimation of Amlodipine Besilate and Bisoprolol Fumarate in Pharmaceutical Preparations - R.B. Kakde, V.H. Kotak and D.L.Kale 14 Development And Validation of Spectrophotometric Method for Determination of Montelukast Sodium in Bulk and Tablet Formulation - Varun Pawar, Lalitha N, Puranik SB, Sanjay Pai P.N, Rao G.K. Gas Chromatographic Determination of Residual Solvents in Pharmaceutical Excipients - S. B. Puranik, Varun R. Pawar, N. Lalitha, P. N. Sanjay Pai, G. K. Rao UV-Spectrophotometry and First Order Derivative Methods for Estimation of Efavirenz in Bulk and Capsules - Charushila H. Bhirud, Atul A. Shirkhedkar, Ravindra A. Fursule and Sanjay J. Surana RP-HPLC Estimation of Eplerenone in Tablets - K. P. Bhusari, P. B. Khedekar, N. D. Amnerkar, S. M. Dhole, V. S. Banode Development and Validation of New RP-HPLC Method with UV-Detection for the Determination of Carvedilol in Human Serum - Mogallapalli L V Setti, Vijaya Ratna J Gas Chromatographic Determination of Methanol and Isopropyl Alcohol Impurities in Herbal Extracts - S. B. Puranik, Varun R. Pawar, Lalitha N, P. N. Sanjay Pai, G. K.Rao Simultaneous Spectrophotometric Estimation of Paracetamol and Tramadol Hydrochloride in Solid Dosage Form - P.G. Yeole, S.J. Wadher, M.P. Puranik, R.O. Ganjiwale First Order Derivative Spectrophotometric Determination of Nebivolol in Bulk and Tablets - Atul A. Shirkhedkar, Prasad M. Bugdane, Sanjay J. Surana 18 21 24 26 28 32 36 39

LIFE is PRECIOUSWE

CARE for the ESSENCE of life

DIVISIONS AT A GLANCE CRITICAL CARE Division COPAS Division

SPECIALTY Division HYGEA Division

ONCOLOGY Division RINON Division

UNITED BIOTECH (P) LIMITEDBagbania, Baddi-Nalagarh Road, Distt. Solan (HP) - 174 101 E-mail : [email protected], Website : www.unitedbiotechindia.com

Application of Oxidants to the Spectrophotometric Determination of Gemifloxacin Mesylate in Pharmaceutical Formulations - Marothu Vamsi Krishna, Dannana Gowri Sankar Simultaneous Estimation of Aceclofenac and Paracetamol in Combined Dosage Form by Two Wavelength Spectrophotometry - Wadher S. J., Momin M. Y., Puranik M. P. and Yeole P. G. Application of UV-Spectrophotometric Method for Estimation of Drotaverine Hydrochloride in Bulk & Tablets - A. A. Shirkhedkar, G. H. Upasani and S.J. Surana Oxidative Coupling, Complex Formation and Internal Salt Formation Reactions for Visible Spectrophotometric Determination of Alfuzosin Hydrochloride in Pharmaceutical Formulations - M. Vamsi Krishna, D. Gowri Sankar Development of Spectrophotometric Analysis Method of Atenolol Gel Formulations - Nandy B. C, Gupta R.N UV-Spectrophotometric Estimation of Nabumetone - M. N. Purohit, Sharad Mohan, Kunal Chokshi, G. V. Pujar Difference Spectroscopic Estimation of Raloxifene in Bulk and Formulations - Patel P. M., Patel R. C. and Patel N. M. Spectrophotometric Methods for the Determination of Rosuvastatin Calcium in Pure Form and in Pharmaceutical Formulations by Using Redox/Complexation Reactions - M. Vamsi Krishna and D. Gowri Sankar A Novel Application of Hydrotropic Solubilization in the Spectrophotometric Estimation of Frusemide in Tablets - R. K. Maheshwari Estimation of Fenoverine Hydrochloride in Pharmaceutical Dosage Forms by RP HPLC - Dr. K. Chitra, K. Sujatha, C. Vinodhini, B. Hareesh, B. Bharath, E. Brahma Reddy and B. Pamula Reddy Evaluation of Marketed Polyherbal Antidiabetic Formulations using Biomarker Charantin - P. M. Patel, N. M. Patel and R. K. Goyal Determination of Cefadroxil in Bulk Powder and its Dosage Forms by Spectrophotometric Method - Haresh M. Patel, Bhanubhai N. Suhagia, Shailesh A. Shah, and Ishwarsinh S. Rathod Reverse Phase HPLC Determination of Celecoxib in Dosage Forms - T. E. G. K. Murthy, Y. A. Chowdary, K. Narendra Kumar, D. Gowri Sankar and B. Durvasa Rao Simultaneous Estimation of Chlorzoxazone and Tramadol Hydrochloride in Tablet Formulation by Two Wavelength Spectrophotometry - M.P. Puranik, Anjali Hirudkar, S.J. Wadher, and P.G. Yeole Spectrophotometric Methods for Quantitative Estimation of Venlafaxine Hydrochloride from Tablet Formulation - S. Pillai and I. Singhvi New Application of Hydrotropic Solubilization in the Spectrophotometric Estimation of Ketoprofen in Tablet Dosage Form - R.K. Maheshwari Spectrophotometric Method for the Estimation of Valdecoxib and Paracetamol from Combined Dosage Form - K. E. V. Nagoji, V. Kiran Kumar, K. Ravi Sankar, V. Venkata Rao, Prafulla Kumar Sahu and M. E. B. Rao Simultaneous estimation of Pantoprazole and Domperidone in Pharmaceuticals Dosage Forms by RP-HPLC - Singh R. A., Arora Saurabh, Kumar Robin, Kumar Dinesh & Agarwal A.K H.P.L.C Method for Simultaneous Estimation of Tannic acid, Gallic acid, Chebulinic acid and Ethyl gallate in Terminalia chebula fruits - K. Jayaram Kumar, S.P. Bhatnagar Simultaneous Estimation of Cetirizine Hydrochloride and Salbutamol Sulphate in Pharmaceuticals dosage forms by RP-HPLC - Dr. R.A. Singh, Robin Kumar, Dinesh Kumar & A.K. Agrawal$

41 44 47 49 52 56 58 61 64 66 68 71 73 75 77 79 82 84 86 88

ANALYTICAL METHOD DEVELOPMENT

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Validated Method Development for Estimation of Atorvastatin Calcium and Fenofibrate in Fixed Dose Combination by HPTLCP. B. Deshpande, G. Shridharan, Libi Anandi, D. Jadhav, M. C. Damle, S. V. Gandhi*

Abstract: A simple high-performance thin-layer chromatographic method has been developed and validated for determination of atorvastatin calcium and fenofibrate in a fixed dose combination. The drugs were separated on aluminum plates precoated with silica gel 60 F254 using chloroformmethanol (8:2 v/v) as solvent system. The drugs were well resolved with Rf values 0.29 and 0.77 for atorvastatin calcium and fenofibrate, respectively. Quantitative analysis was performed by densitometric scanning at 285 nm. The method was validated for linearity, accuracy, precision, specificity and robustness. The calibration plot was linear over the ranges 2001000 and 3201600 ng/band for atorvastatin calcium and fenofibrate, respectively. The method was successfully applied to the analysis of drugs in a pharmaceutical formulation.

IntroductionAtorvastatin calcium is a selective competitive inhibitor of the enzyme HMG-CoA reductase that catalyses conversion of HMGCoA to mevalonate, an important rate-limiting steps in cholesterol biosynthesis.1 Fenofibrate is a lipid lowering agent.2 Literature survey reveals that few HPLC and HPTLC methods have been reported for estimation of atorvastatin calcium3,4 and fenofibrate5,6 in combination with other drugs. One HPTLC method reported by Chaudhari et. al.7 for simultaneous estimation of atorvastatin calcium and fenofibrate used toluene:methanol: acetic acid (08:02:02 v/v/v) as solvent system. The limitation of this method primarily is high Rf value for fenofibrate (0.88) which makes precise densitometric evaluation rather difficult due to interference by solvent front.

fenofibrate were purchased from local market. All chemicals used were of analytical-grade. Preparation of Standard and Sample Solutions Atorvastatin calcium (10 mg) and fenofibrate (160 mg) were weighed separately, transferred to separate 10 ml volumetric flasks and dissolved in 10 ml of methanol. Stock solutions were further diluted with methanol to furnish working standard solution of concentration 100 ng/l for atorvastatin calcium and 160 ng/l for fenofibrate. For analysis of the tablet dosage form, the powdered sample equivalent to 10 mg atorvastatin calcium (160 mg fenofibrate) was accurately weighed and dissolved in 10 ml methanol. This solution was filtered and 1 ml of filtrate was further diluted to 10 ml with methanol to furnish solution of concentration 100 ng/l for atorvastatin calcium determination (Test Solution I). The 1 ml of second dilution was further diluted to 10 ml to furnish solution of concentration 160 ng/l of fenofibrate (Test Solution II). TLC Method and Chromatographic Conditions TLC was performed on 20 cm 20 cm aluminium-backed HPTLC plates coated with 0.2 mm layers of silica gel 60 F254 (E. Merck, Germany). Before use the plates were pre-washed with

ExperimentalChemicals and Reagents Pharmaceutical grade atorvastatin calcium and fenofibrate were obtained as gifts from Mepro Pharmaceuticals Pvt. Ltd., Wadhawan (Gujarat) and A TO Z Pharmaceuticals Pvt. Ltd., Chennai (Tamilnadu), respectively. Fixed dose combination tablets containing 10 mg of atorvastatin calcium and 160 mg of

Department of Pharmaceutical Analysis, AISSMS College of Pharmacy, Kennedy Road, Pune. *Author for correspondence e-mail: [email protected]

3

ANALYTICAL METHOD DEVELOPMENTmethanol, activated in an oven at 105OC for 20 min, then left to cool to room temperature. Solutions were applied to the plates, as 8 mm bands by means of a Camag Linomat IV semi-automatic sample applicator fitted with a 100-?l Hamilton syringe. Plates were then developed with 20 ml chloroformmethanol 8:2 (v/v) as mobile phase, in a 20 cm 20 cm Camag twin-trough chamber previously saturated for 20 min. The development distance was 15 cm (development time 20 min). After development the plates were removed from the chamber, dried in air and densitometric scanning at 285 nm in reflectance mode was performed with a Camag TLC Scanner-3 equipped with winCATs software version 1.4.2 incorporating track-position optimization. The slit dimensions were 6.00 mm 0.45 mm. Assay of the Marketed Formulation For assay of marketed formulation fixed volumes of test solution I and II (4 l for atorvastatin calcium and fenofibrate each) were applied to the plate which was then developed as described above. The amount of atorvastatin calcium and fenofibrate was determined from the standard calibration curve and the amount of each drug in sample was calculated. Validation of Method The method was validated in accordance with ICH guidelines for linearity, accuracy, specificity, intra-day and inter-day precision as well as robustness. For preparation of calibration plots aliquots of working standard solutions of atorvastatin calcium (100 ng/l) and fenofibrate (160 ng/l) were applied to the plates which were then chromatographed and processed as described under chromatographic conditions. To study the accuracy of the method, recovery studies were carried out by addition of standard drug solution to pre-analyzed sample solution at three different levels 50, 100, and 150 %. To study intra-day variation, six mixed standard solutions containing atorvastatin calcium (400 ng/band) and fenofibrate (640 ng/band) were prepared and applied to the plates. All solutions were analysed on the same day to record any intra-day variation in the results. To study inter-day variation, analysis of three mixed standard solutions of the same concentration was performed on three different days. To confirm the specificity of the method, solutions of atorvastatin calcium and fenofibrate and the extract from the tablet dosage form were applied to a TLC plate, plate was developed and scanned as described above. The effect of small, deliberate variation of the analytical conditions on the peak areas of the drugs was examined. Factors varied were volume of mobile phase ( 0.5 %), time from application to development (0, 10, 20, and 30 min) and

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from development to scanning (0, 30, 60, and 90 min). One factor at a time was changed to study the effect. The robustness of the method was checked at amount of 400 and 640 ng/band for atorvastatin calcium and fenofibrate, respectively.

Results and DiscussionAs the HPTLC method reported by chaudhari et. al. for simultaneous HPTLC estimation of atorvastatin calcium and fenofibrate has very high Rf values particularly for fenofibrate (0.88), the objective of the current study was to develop a more accurate, precise, versatile, speedy and cost-effective HPTLC technique for determination of atorvastatin calcium and fenofibrate in fixed dose combination. Different mobile phases containing different proportions of chloroform, toluene, methanol, acetone, ethyl acetate, n-butanol and triethylamine were examined. The mixture of chloroformmethanol 8:2 (v/v) enabled satisfactory resolution of atorvastatin calcium and fenofibrate with good peak shape, Rf values of 0.29 0.019 and 0.77 0.025, respectively (Figure 1).

Standard calibration plots were linear over the range 200 1000 ng/band for atorvastatin calcium and 320 -1600 ng/band for fenofibrate with high correlation coefficient. Excipients present in the formulation did not interfere with the peaks of atorvastatin calcium and fenofibrate. The spectra of atorvastatin calcium and fenofibrate extracted from the tablets were also compared with the spectra obtained from atorvastatin calcium and fenofibrate standards and correlation was found to be good. The recovery was found to be 98.59-99.05 % for atorvastatin calcium and 99.15 99.32 % for fenofibrate. Intra-day variation, as RSD (%) was 0.640 for atorvastatin calcium and 0.412 for fenofibrate, with standard error 0.216 and 0.146, respectively. Interday variation, as RSD (%), was 0.710 for atorvastatin calcium and 0.580 for fenofibrate, with standard error 0.354 and 0.762, respectively. The method was also evaluated by assay of commerciallywww.kppub.com

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ANALYTICAL METHOD DEVELOPMENTavailable tablets containing atorvastatin calcium and fenofibrate. Six replicate analyses were performed on accurately weighed amounts of the tablets. The assay (%) was found to be 98.30 0.29 for atorvastatin calcium and 99.10 0.23 (Mean S.D.) for fenofibrate. Study of the robustness of the method revealed that peak areas were unaffected (RSD < 2 %) by small changes of the operating conditions.

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Bothara, Principal, AISSMS College of Pharmacy, Pune, India for providing infrastructure facilities to carry out this research work.

References1. The Merck Index, 14th edition, Edited by Maryadele J. O'Neil, Merck Research Laboratories, Division of Merck and Con., Inc., Whitehouse Station, NJ, 2006, 864. Remingtons- The Science and Practice of Pharmacy, 21st Edition, Vol. II, Edited by Beringer P., Mack Publishing Co., Easton, PA, 2005, 1368. Erturk, S., Sevinc, E., Erosy, L. and Ficicioglu, S., J. Pharm. Biomed. Anal., 2003, 33, 1017. Rajeswari, K., Sankar, G. and Seshagirirao, J., Indian J. Pharm. Sci., 2006, 68, 275. Rani, S., Nivsarkar, M., Rathod, R., Guttikar, S. And Padh, A., Indian J. Pharm. Sci., 2005, 67, 297. EIGindy, A., Emara, S., Mesbah, M. and Hadad, G., Farmaco., 2005, 60, 425. Chaudhari, B. G. and Patel, N. M., Indian Drugs, 2007, 44, 378.

2. 3. 4. 5. 6. 7.

ConclusionThis validated HPTLC method can be used for routine analysis of atorvastatin calcium and fenofibrate in combined tablet dosage forms.

AcknowledgementsAuthors are thankful to Mepro Pharmaceuticals Pvt. Ltd., Wadhwan (Gujarat) and A TO Z Pharmaceuticals Pvt. Ltd. (Chennai) for providing gift samples of atorvastatin calcium and fenofibrate, respectively and are also thankful to Dr. K. G.

Source: The Pharma Review, May 2009

www.kppub.com

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ANALYTICAL METHOD DEVELOPMENT

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Simultaneous High Performance Liquid Chromatographic Estimation of AmphotericinB in Dry Injection Dosage FormJ.P. Mall*, P.C. Patel And Dr. J.N. Verma

Abstract: Simple, accurate and reproducible High Performance Liquid Chromatographic method, requiring no prior Instrumental methods have been developed for the estimation of Amphotericin B in Amphotericin B injection mentioned in I.P.2007,Volume2,Page No.727and728/USP 2007,Volume2,Page No.1410. In both monograph method of content estimation is determind by Microbiological assay of Antibiotics.As instrumental analysis is more accurate as well as less time consuming. Liquid Chromatographic method for the estimation of Amphotericin B was developed using Reversed phase H.P.L.C. having ultra violet detector and separation was carried out on Column C18(25X4.6mm,5Micron), using Water 486, mode by direct injection.. The retention time for standard and test was found to be 6.4 minutes.The method was validated according to ICH guidelines.The method described is simple, sensitive, reliable and reproducible for the quantitative estimation of active ingredient in dosage forms and their levels are found to be within the ICH limits.

IntroductionAmphotericinB is Antifungal agent, official in Indian Pharmacopoeia and United State Pharmacopoeia, chemically it is (3-amino-3,6-di deoxy-b-D-mannopyranosyloxy)-16-Carboxy3,5,8,9,11,13,15,35-octahydroxy-34,36-dimethyl-13,17-epoxyoctatviaconta-20,22,24,26,28,30,32-heptaen-37-olide and other antifungal polyenes produced by the growth of certain strains of Streptomyces nododus or by any other means. The review of literature revealed that no instrumental or chemical method is as yet reported for the simultaneous estimation of the drugs in dosage forms. This paper describes simple, rapid, accurate and reproducible method for determination of drugs in Injection dosage form.

Reagent: Acetonitrile and methanol were used of H.P.L.C. grade and Sodium dihydrogen ortho phosphate were used of A.R.grade.Other chemicals i.e. dimethylsulfoxide were used of A.R.grade.Mobile phase were prepared with distilled water and degassed in sonicator subject to filteration using Whatman filter paper No.41.In case of Standard and Sample preparation, it was filtered using 13 mm membrane filter of 0.2 micron pore size.

ProcedureStandard Preperation: Dissolve 50 mg. of Amphotericin B Working Standard in 50 ml. of dimethyl sulfoxide. Transfer 2.5 ml.from this prepared solution to another 50 ml. Volumetric flask, add 10 ml. dimethyl sulfoxide and make volume up to the mark with mobile phase, mix well. Test Preperation: As Amphotericin B for injection is a sterile freeze dried mixture of Amphotericin B and deoxycholate sodium with buffering agent and is filled in a sealed container. Dissolve content of vial using 20 ml.of distilled water. Transfer 1 ml.from this prepared solution to 50 ml. volumetric flask, add 10 ml.of dimethyl sulfoxide and make up volume to 50 ml. with mobile phase.

ExperimentInstrument: Water 486 Column C18 (25X4.6 mm, 5 Micron) Wavelength UV at 405 Flow Rate: 1.0 ml/ Minute

Lifecare Innovations. *Author for correspondence E-mail: [email protected]

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ANALYTICAL METHOD DEVELOPMENTBlank: Transfer 10 ml. of dimethyl sulfoxide to 50 ml.volumetric flask and make up volume to 50 ml. with mobile phase. Final concentration of Standard and Sample preparation is about 50 mcg/ml. Claim: Each vial contains Amphotericin B- 50 mg. Mobile Phase: Mixture of Acetonitrile-Methanol and 0.01M Sodium dihydrogen orthophosphate in the ratio of 41: 10: 49 were used. Column: C18 (25X4.6 mm, 5 Micron) Wavelength: U V at 405 Flow Rate: 1.0ml/ Minute Both the dilutions of Standard and Sample preparation were injected in reversed phase H.P.L.C. having same condition and retention time for standard and test were observed same i.e. 6.4Minutes. Calculation:Sample Area Standard Area X Standard dilution Sample dilution Potency of Std. 100

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Table 2 Results of Analysis of commercial injection by Microbiological Assay Method Sample AmphotericinB for Injection Label Claim mg/ml % of Label Claim Estimated 50.0 mg/ml 50.02mg/Vial(100.04%)

Results of analysis by both method were found almost same.

Recovery StudiesTo study the accuracy, reproducibility and precision of the above proposed method , recovery studies were carried out by addition of Standard drug to pre analyzed sample. Results of recovery studies were found to be satisfactory.S.N. Concentration of added Amount recovered % Recovery amount of drug 1. 2. 3.0.53

5.0 mg. 10.0 mg. 15.0 mg.

4.90 mg. 9.94 mg. 15.02mg.

98.0 99.4 100.1

Au

0.244.75. 4.93

X

X Av.filled wt.2.12

2.50, 2.71 2.88, 2.98 3.45, 3.73

Procedure for Analysis of Powder for InjectionPowder for Injection are sterile, solid substances, freeze-dried materials which are distributed in their final containers and which, when shaken with the prescribed volume of the appropriate sterile liquid, rapidly form clear and practically particle free solutions. In case of powder for injection 10 vials are selected at randomly and perform the content uniformity as per method described.Table1 Sample AmphotericinB for Injection Label Claim mg/Vial % of Label Claim Estimated 50.0 mg/ml 50.15 mg/Vial(100.3%)-0.06 0.00

7.68 8.03 8.26

5.59

9.83

Min

19.66

Typical Chromatogram of HPLC

Results and DiscussionThe low value of Standard deviation and recovery was found close to 100%(limit98% to 101%), indicates the reproducibility and accuracy of the method. The method employing is very simple as well as rapid and accurate. and can be employed for routine analysis in this dosage forms using simple instrumentation. So this method can be employed for routine analysis in Quality Control laboratories.

Results of Analysis of commercial injection by proposed methodThis is mean of five determination. In above case of Table1 standard deviation of peak area were observed about 1%, well within the standard limit of 2%. Simultaneous determination of drugs in Injection by Microbiological Assay method were observed. This is mean of five determination.

AcknowledgementsAuthors are grateful to Managing Director-Lifecare Innovations for his co-operation

References1. 2. Antimicrob Agents Chemother 1986 April;29(4):584-588.PMC 180446 J.ChromatogrB Biomed Sci Appl.2001 Sep 5,760(2):307-13.Related Articles Links.

Source: The Pharma Review, March 2009www.kppub.com

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ANALYTICAL METHOD DEVELOPMENT

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Stress Degradation Studies on Ceftazidime Sodium and Development of a Validated Stability-Indicating HPLC AssayLalitha N*, S.B.Puranik, Sanjay Pai P.N, Rao G.K.

Abstract: Ceftazidime was subjected to different ICH prescribed stress conditions. The drug was found more stable under neutral, photolytic, 3% oxidative and solid state conditions, while it was found to be less stable under acidic, alkaline and 30% oxidative conditions. A stability-indicating HPLC method was developed for analysis of the drug in the presence of the degradation products. It involved a C-18 column and a mobile phase composed of phosphate buffer pH 6.8 and methanol (10:3), which was pumped through the column in isocratic mode. The detection was carried out at 254 nm. The method was validated for linearity, range, precision, accuracy, specificity, selectivity and intermediate precision.

1. IntroductionThe parent drug stability test guideline Q1A (R2) issued by International Conference on Harmonization (ICH)1 suggests that stress studies should be carried out on a drug to establish its inherent stability characteristics, leading to identification of degradation products and hence supporting the suitability of the proposed analytical procedures. It also requires that analytical test procedures for stability samples should be stability-indicating and they should be fully validated. Accordingly, the aims of the present study were to establish inherent stability of ceftazidime through stress studies under a variety of ICH recommended test conditions1 and,2 and to develop a stability-indicating assay.3 The drug is Pyridinium, 1-[[7-[[(2amino-4-thiazolyl)[(1-carboxy-1methyle-thoxy)imino]acetyl] amino]-2-carboxy-8-oxo-5thia-1-azabicyclo[4.2.0]oct-2-en-3yl]methyl]-,hydroxide,innersalt,pentahydrate,[6R-[6a,7b(Z)]] (Fig. 1).4,5 It is a white powder that is soluble in water and practically insoluble in alcohol. It is a semi synthetic 2nd generation cephalosporin with broad-spectrum, beta-lactamaseresistant available in reconstituted formulation. The literature survey reveals that methods are available for the mechanism of ceftazidime degradation in aqueous solutions,6 stability of ceftazidime pentahydrate in medicinal preparation biotium and ceftim,7 reversed-phase HPLC method for determination of the drug in pharmaceutical dosage forms,8

Fig. 1: Structural formula of Ceftazidime O SH2N N NH N H H O O N

ON+

S

O

H 3C H3C

OH O

Determination of Ceftazidime by Thin-Layer Chromatography and Densitometry,9 First-derivative spectrophotometric and LC determination of ceftazidime and cefadroxil in urine,10 Analysis of antibiotics in urine and wipe samples from environmental and biological monitoring comparison of HPLC with UV, single MSand tandem MS-detection.11 Accordingly, a stability-indicating method was developed, which could separate various degradation products.

2. Experimental2.1. Materials Ceftazidime sodium was supplied by Karnataka Antibiotics Pharmaceuticals Ltd, Bangalore, India and used as such. Methanol (HPLC grade) was purchased from Qualigen Fine Chemicals, Mumbai, India. The other chemicals and solvents used in the studies were of analytical grade. Ultra-pure water, obtained from Millipak 0.22 m water purification systems.

Al-Ameen College of Pharmacy, Near Lalbagh Main Gate, Bangalore. *Author for correspondence E-mail: [email protected]

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ANALYTICAL METHOD DEVELOPMENT2.2. Instrumentation Precision water baths equipped with MV controller (Julabo, Seelbach, Germany) were used for hydrolytic studies. Stability studies were carried out in humidity chamber (KBF 760, WTB Binder, Tuttlingen, Germany) and photo stability studies were carried out in photostability chamber designed Analytical Technologies Ltd. Bangalore using D65 and UV lamps. Thermal stability studies were performed in a dry air oven (NSW Limited, New Delhi, India). The mobile phase was degassed by Mark Ultrasonic sonicator. The HPLC system consisted of isocratic pumps (LC-10ALVP) and UV-visible dual-wavelength detector (SPD-10AVP), Winchrome-VP software (all from Shimadzu, Kyoto, Japan). The chromatographic separations were carried out on SS Accurasil (SGE) C-18 column of 250 mm 4.6 mm i.d. with particle size of 5 m. Additionally, intermediate precision studies were also carried out on Wakosil (SGE) C-18 column (250 mm 4.6 mm i.d., particle size 5 m). 2.3. Degradation studies All stress decomposition studies were performed at an initial drug concentration of 1 mg ml-1 in phosphate buffer (pH 6.8) and methanol (10:3). Acid hydrolysis was performed in 0.1M HCl at 80C for 30 min. The study in alkaline condition was carried out in 0.1 M NaOH at 80C for 10 min. For the study in neutral condition, drug dissolved in water was heated at 80C for 180 min. Oxidative studies were carried out at room temperature in 3% and 30% hydrogen peroxide for 24 h and 30 min respectively. Photodegradation studies were performed in water and in 0.1 M HCl. The solutions were exposed to D65 (60,000-70,000 lux) for 24 h. Suitable controls were kept under the dark. Additionally, the drug powder was exposed to dry heat at 50C for 8 d. Samples were withdrawn at appropriate time intervals and subjected to HPLC analysis after suitable dilution. 2.4. Separation studies HPLC studies were carried out first on all reaction solutions individually and then on a mixture of those solutions in which decomposition was observed. Separations were achieved by isocratic elution using phosphate buffer (pH 6.8) and methanol in the ratio of 10:3 as the mobile phase. The mobile phase was filtered through 0.22 m nylon membrane and degassed before use. The injection volume was 100 L and the mobile phase flow rate was kept constant at 0.5 ml min-1. The analyses were carried out at 254 nm. 2.5. Validation of the method 2.5.1. Linearity and range

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A stock solution of the drug was prepared at strength of 1 mg mL-1. It was diluted to prepare solutions containing 0.5 100 g mL-1 of the drug. The solutions were injected in triplicate into the HPLC column, keeping the injection volume constant (100 L) 2.5.2. Precision Six injections, of three different concentrations (5, 10 and 20 g mL-1), were given on the same day and the values of relative standard deviation (R.S.D.) were calculated to determine intraday precision. These studies were also repeated on different days to determine inter-day precision. 2.5.3. Accuracy Accuracy was evaluated by fortifying a mixture of degraded solutions with four known concentrations of the drug. The recovery of the added drug was determined. 2.5.4. Specificity and selectivity The specificity of the method was established through study of resolution factors of the drug peak from the nearest resolving peak, and also among all other peaks. 2.5.5. Intermediate precision The intermediate precision was established through a study on a different chromatographic system using a different column.

3. Results and discussion3.1. Degradation behavior HPLC studies on Ceftazidime under different stress conditions suggested the following degradation behavior: 3.1.1. Acidic condition The drug gradually decreased with time on heating at 80C in 0.1 M HCl, forming degradation products at RT 1.83 and 3.1 in 20 min. At the end of 30 min the drug was completely degraded. 3.1.2. Neutral (water) condition Upon heating the drug solution in water at 80C for 30 min, almost 20% of drug was degraded. At the end of 180 min, complete degradation of the drug was observed with the corresponding rise in the major degradation peaks at RT 1.81, 2.64 and 3.04 min. 3.1.3. Degradation in alkali The reaction in 0.1 M NaOH at 80C was so fast that whole of the drug was degraded in 10 min. Drug degradation was associated with rise in a major degradation product at RT 1.92, 2.6 and 3 min.

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ANALYTICAL METHOD DEVELOPMENT3.1.4. Oxidative conditions In 3% H2O2, the drug was degraded within 12 h and completely degraded at the end of 24 h. Drug was degraded completely at the end of 30 min in 30% H2O2. The oxidative degradation of drug was associated with rise in major degradation products at RT 1.9 and 3.14 min. 3.1.5. Photolytic conditions Major degradation products were observed after exposure of drug solution in 0.1 M HCl to D65 for 12 h at RT 1.8 and 3.1 were formed. The nature of degradation in light and dark was found to be similar, indicating that light had no effect on the degradation of the drug in acid. On the other hand, the samples in water degraded under D65 for 24 h to a major product at RT 2.7, along with minor degradation products at RT 1.87 and 3.14. Corresponding rate of degradation in dark was much slower. 3.1.6. Solid-state study The solid-state studies showed that ceftazidime was stable to the effect of temperature. When the drug powder was exposed to dry heat at 50 C for 8 d, 20.85% drug was degraded without giving any degradation product. 3.2. Development and optimization of the stability-indicating method The method was optimized to separate major degradation products formed under various conditions. Resolution was also checked on mixture of the degradation solutions to confirm the separation behavior. The resulting chromatogram is shown in Fig. 2. It indicates that the isocratic method was successful in separation of drug and all chromophoric degradation products. 3.3. Validation of developed stability-indicating method The response for the drug was strictly linear in the concentration range between 5 and 100 g mL-1. The mean ( %R.S.D.) values of slope, intercept and correlation coefficient were 22662 (1.13), 28804 (1.805) and 0.9977 (0.015), respectively. The data obtained from precision experiments are given in Table 1 for intra-and inter-day precision studies. The %R.S.D. values for intra-day precision study were 0.998) for all the three methods. The proposed methods were also evaluated by the assay of commercially available tablets containing OM and HTZ (n = 6). The % assay represented as mean S.D. (% RSD) of commercial formulation by three methods was found to be 101.62 0.401. (0.394), 100.14 0.507. (0.506), 100.49 0.018. (0.017) and 100.85 0.854. (0.847), 101.61 0.054. (0.053), 100.23 0.489 (0.488) for OM and HTZ by method I, II and III respectively For recovery study different volumes of standard stock solution were added to the fixed volume of sample solution at 50 %, 100 % and 150 % level. The total amount of drug added was determined by proposed methods, results of which are shown in Table 1.

Abs 10.5 0 200

OM HTZ

250

300 Wave length (nm)

350

400

Fig. 1: Zero order overlain spectra of OM (16g/mL) and HTZ (16 g/mL)

Absorbance Correction Method (Method II) This method involves absorbance correction for OM determination by subtracting absorbance of HTZ from total absorbance of sample at 256 nm (max of OM). Since at 317.4 nm OM shows nearly zero absorbance; HTZ concentration is directly determined from absorbance of sample at this

ConclusionsThe validated spectrophotometric methods can be used for routine determination of OM and HTZ in combined tablet dosage form.www.kppub.com

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ANALYTICAL METHOD DEVELOPMENTTable 1: Recovery studies of OM and HTZ Drug Conc. of drug added Method Ia

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2.

Method II % RSD % Recovery S.D.a

Method III % % Recovery RSD S.D.a

http://en.wikipedia.org/wiki/Hydrochlorothiazide. accessed on 28.07.2007 Celebier, M. and Altinoz, S., Pharmazie, 2007, 62, 419. Yu, Y., Hou, Y.N., Liu, J.F. and Wang.J., J. Chin. Pharm, 2006, 41, 1592. Liu, D., Hu, P., Matsushima, N., Li, X., Li, L. and Jiang, J. J. Chromatogr. B., 2007,856, 190. Lande, N. R., Shetkar,B. M., Kadam, S. S. and Daneshwar, S. R., Indian drug, 2000, 37, 574. Suhagia, B.N., Shah, R.R. and Patel, D.M., Indian J. Pharm. Sci., 2005, 67, 37. Farthing, D., Fakhry, I., Elizabeht, B.D., Ripley, D.S., J. Pharm. Biomed. Anal., 1998, 17, 1455.

3.

g/ % % Recovery ml Level S.D. OM 4 8 12 HTZ 2.5 5 12.5a

% RSD

4. 5. 6. 7. 8.

50

99.640.826

0.829 100.290.302 0.301 100.660.161 0.160 0.632 100.460.532 0.530 100.350.893 0.890

100 99.580.629 50

150 100.550.679 0.676 100.310.211 0.211 100.690.980 0.974 100.610.826 0.821 101.610.982 0.975 100.230.718 0.716 0.612 100.540.597 0.594 99.960.227 0.227 0.953 100 99.280.607

150 100.150.404 0.404 100.630.195 0.194 99.690.950

average of three determinations

AcknowledgmentThe authors are thankful to Micro Labs Ltd. for providing the pure drug samples. Thanks are also extended to Principal, Dr. K. G. Bothara, AISSMS College of Pharmacy for providing infrastructure facilities.

9. Zendelovska, D., Stafilov, T. and Milosevski, P., Biomed. Chromatogram., 2004, 18, 71. 10. Shah, N.J., Suhagia, B.N., Shah, R.R.and Patel, D.M., Indian J. Pharm. Sci., 2007, 69, 240. 11. Razak, O.A. J. Pharm. Biomed. Anal., 2004, 34,433. 12. Belal, F., Al-Zaagi, I.A., Gadkariem, E.A. and Abounassif, M.A. J. Pharm. Biomed. Anal., 2001, 24 , 335. 13. ICH Harmonised Tripartite Guideline (Nov. 2005) Validation of Analytical Procedures: Text and Methodology Q2 (R1).

References1. http://www.pharmmarketing.com/bulk_drug_eng.htm. accessed on 30.07.2007

Source: The Pharma Review, December 2008

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High Performance Thin Layer Chromatographic Method for Simultaneous Estimation of Amlodipine Besilate and Bisoprolol Fumarate in Pharmaceutical PreparationsR.B. Kakde*, V.H. Kotak and D.L.Kale

Abstract: A novel analytical method for simultaneous determination of amlodipine besilate and bisoprolol fumarate in their combined dosage form has been developed by HPTLC procedure, using ethyl acetate : methanol : ammonia (6:0.5:0.5 v/v/v) as mobile phase and Merck HPTLC plate precoated 60 F254 silica gel on aluminum sheet as stationary phase. Detection was carried out densitrometrically using UV detector at 229 nm. The retention factor (RF) of amlodipine besilate and bisoprolol fumarate was 0.390.02 and 0.520.02 respectively. The linear regression analysis data was used for the regression line in the range of 500-1000 ng/spot for both amlodipine besilate and bisoprolol fumarate with correlation coefficient value close to 1.0. The developed method was validated using various validation parameters such as accuracy, precision, specificity, limit of detection, limit of quantitation and ruggedness, which make method as choice for routine quality control analysis for simultaneous estimation of amlodipine besilate and bisoprolol fumarate in their combined formulation.

IntroductionAmlodipine (a dihydropyridine) is a calcium-channel blocker. It is given as besilate and used in the management of hypertension and angina pectoris. It is chemically 3-ethyl-5-methyl-2-(2aminoethoxymethyl)-4-(2-chlorophenyl)-1,4-dihydro-6methylpyridine-3,5-dicarboxylate monobenzenesulphonate. It is official in BP.1 Bisoprolol is a cardioselective beta blocker. It is given as the fumarate in the management of hypertension and angina pectoris. Chemically it is ()-1-[4-[[2-(1-methylethoxy) ethoxy] methyl] phenoxyl-3-[(1-methyl ethyl) amino]-2-propanol.It is official in USP.2 Literature survey reveals the availability of several UV,3-6 HPLC7-10 and HPTLC10 methods for estimation of amlodipine besilate (AMLO) as alone or in combination with other drugs and bisoprolol fumarate (BISO) can be determined by UV,11 RPHPLC,12 HPTLC13 in combination with hydrochlorthiazide and RPHPLC14 as alone is reported. Present work emphasizes on the quantitative estimation of amlodipine besilate and bisoprolol fumarate in their combined dosage form by HPTLC.

ExperimentalPreparation of Standard Solutions Standard stock solution was prepared by dissolving 69.34 mg of amlodipine besilate (equivalent to 50 mg of amlodipine) and 50 mg bisoprolol fumarate in 50 mL of methanol. Standard solution was prepared by diluting 5 mL of this solution to 50-mL with methanol to get final concentration of 100 g mL-1 for both the drugs. Preparation of Sample Solution Twenty tablets were accurately weighed and average weight was calculated. The tablets were then crushed to obtain fine powder; an accurately weighed quantity of tablet powder equivalent to about 5 mg amlodipine was transferred to 50 mL volumetric flask and shaken with 15-20 mL methanol for 30 minutes for dissolving active ingredients completely, then volume was made up to the mark with methanol, the solution was mixed and filtered through Whatman Grade I filter paper. The concentration of final solution was 100 g mL-1 for both drugs.

University Department of Pharmaceutical Sciences, RTM Nagpur University, Nagpur. *Author for correspondence E-mail: [email protected]

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ANALYTICAL METHOD DEVELOPMENTThin-Layer Chromatographic Analysis The standard solution was applied in the form of bands of 4 mm width with space of 4 mm on the precoated silica gel aluminium plate 60 F254 (10cm10cm) in the range of 1-10 L with the help of microliter syringe using LINOMAT-IV automatic sample applicator. The mobile phase consists of methanol: ethyl acetate: ammonia (0.5:6:0.5, v/v/v). Linear ascending development was carried out in 10 cm 10 cm twin trough glass chamber (Camag, Muttenz, Switzerland). The optimized chamber saturation time for mobile phase was 20 minutes at room temperature (25 2OC) at relative humidity of 60 5%. The length of chromatogram run was 70 mm. Subsequent to the development; TLC plates were dried in a current of dry air with the help of an air dryer. Densitometric scanning was performed on Camag TLC scanner III in reflectance-absorbance mode at 229 nm (Fig.1) for all measurements and operated by WINCATS 4.0 software.100.0 [AU] 80.0 70.0 60.0 50.0 40.0 30.0 20.0 10.0 0.0 100.0 [AU] 80.0 70.0

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For specificity studies sample solutions were prepared by exposing an accurately weighed quantity of tablet powder equivalent to about 5 mg of amlodipine to various stress conditions for 24 hrs like at 50OC after addition of 1.0 mL of 0.1 N HCl (acid), at 50OC after addition of 1.0 mL of 0.1 N NaOH (alkali), at 50OC after addition of 1.0 mL of 3.0% H2O2 (oxide), at 60OC and in UV-cabinet at 265 nm.

Result and DiscussionAnalytical Parameters and Validation The method was validated for accuracy, precision, specificity and ruggedness. Linearity was determined at different concentrations, amlodipine besilate showed good correlation coefficient in the concentration range of 500-1000 ng/spot (r = 0.999 and 0.999 by height and area respectively) and bisoprolol fumarate in the range of 5001000 ng/spot (r = 0.997 and 0.998 by height and area respectively).The correlation coefficient value is close to 1.0 shows that amlodipine besilate and bisoprolol fumarate follows linearity with varying concentration. Limit of detection (LOD) and Limit of quantitation (LOQ) were determined by standard deviation of response and slope of calibration curve as per ICH15 guidelines. System reproducibility was determined by five replicate applications and five times measurement of a laboratory mixture at the analytical concentration. The reproducibility of sample with respect to height and area for active compounds were expressed in terms of S.D. and %R.S.D. The spots at RF 0.39 (Amlodipine besilate) and 0.52 (Bisoprolol fumarate) were observed in the densitograms of the drug samples of tablets (Fig.2). There was no interference from the common excipients present in tablets.600 500

Detector Response

BISO AMLO

60.0 50.0 40.0 30.0 20.0 10.0 0.0

200.0 229 250.0

300.0 Wave length (nm)

[nm]

400.0

Fig. 1: Overlain UV Spectra of Amlodipine Besilate and Bisoprold Fumarate for selection of suitable wave length (229nm) by scanning.

The source of radiation utilized was deuterium lamp. Evaluation was done via peak height and peak area. The linear regression analysis data was used for the regression line in the range of 500-1000 ng/spot for both the drugs. Precision of the method was determined by analyzing the marketed formulations. Accuracy of the proposed method was ascertained by carrying out recovery studies by standard addition method. To fixed amount of market formulation different concentrations of mixed pure drug solution of amlodipine besilate and bisoprolol fumarate were added. The sample solutions containing market formulation and pure drug solution were in the range of 80-120% of the concentration mentioned under sample preparation for tablets.

Detector Response

400 300 200 100 0 0.01 0.21 0.41 0.61 0.81 1.101

A

B

Rf valve A - Amlodipine besilate B - Bisoprolol fumarate Fig. 2: Typical densitograms of AB and BFwww.kppub.com

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ANALYTICAL METHOD DEVELOPMENTThe results of recovery studies as evaluated by height and area were found to be close to 100% which signifies the accuracy of the method. Specificity studies results reveals that both the drugs degraded in presence of peroxide and this was confirmed by decrease in height and area of both the drugs, when compared with the spot of standard solution but there was no change in RF value. Ruggedness was performed under three different conditions different days, different analysts and intra day. The results show the %RSD values Method A > Method B > Method D. In conclusion the proposed spectrophotometric methods for the estimation of GFX can be used for the routine quality control of the drug in bulk as well as in pharmaceutical formulations.www.kppub.com

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ANALYTICAL METHOD DEVELOPMENTTable 1: Optical, Regression Characteristics of the Proposed Methods for GFXParameter gmax (nm) Method A 510 Method B 520 Method C 760 Method D 825

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References1.

Oh J I, Pack M J, Ahn M Y, Kim CY, Hong C Y, Kim I C and Kwak J H (1996) In vitro and in vivo evaluations of LB Beer's law limits (g ml-1) 3.0 15.0 4.0 20.0 2.0 - 10.0 6.0 - 30.0 20304, a new fluoronaphthyridone. Antimicrob Agents -1 Chemother. 40; 1564-1568. Detection limits (g ml ) 0.102 0.097 0.053 0.216 2. Cormican M G and Jones R N (1997) Antimicrobial activity Molar absorptivity (L mole-1 cm-1) 2.70 x 104 2.06 x 104 4.27 x 104 1.38 x 104 and spectrum of LB 20304, a new fluoronaphthyridone. Sandell's sensitivity 0.017 0.0023 0.011 0.035 Antimicrob Agents Chemother. 41; 204-211. (g cm-2 / 0.001 absorbance unit) 3. Hohl A F, Frei R, Ponter V, Von graevenitz A, Knapp C, Washington J, Johnson D and Jones R N (1998) Regression equation (Y = a + bC) -2 -2 -2 -2 International multicenter investigation of LB 20304, a new Slope (b) 5.5 x 10 4.2 x 10 8.7 x 10 2.8 x 10 fluoronaphthyridone. Clin Microbiol Infect. 4; 280-284. -3 -3 -3 -3 Standard deviation of slope (Sb) 0.19 x 10 0.10 x 10 0.24 x 10 0.10 x 10 4. Doyle E, Fowles S E, Mc Donnell D F, Mc Carthy and White S A (2000) Rapid determination of gemifloxacin in Intercept (a) 1.3 x 10-3 0.40 x 10-3 -0.50 x 10-3 -0.40 x 10-3 human plasma by high-performance liquid -3 -3 -3 -3 Standard deviation of intercept (Sa) 1.91 x 10 1.38 x 10 1.57 x 10 2.06 x 10 chromatography-tandem mass spectrometry. Journal of chromatography B. 746; 191-198. Standard error of estimation (Se) 1.82 x 10-3 1.32 x 10-3 1.49 x 10-3 1.97 x 10-3 5. Ramji J V, Austin N E, Boyle G W, Chalker M H, Duncan G, Correlation coefficient (r) 0.9999 0.9999 0.9999 0.9999 Fairless A J, Hollis F J, Mc Donnell D F, Musick T J and a Relative standard deviation (%) 0.077 0.109 0.107 0.108 Shardlow P C(2001) The disposition of gemifloxacin,a new fluoroquinolone antibiotic in rats and dogs. Drug % Range of error(Confidence limits)a Metabolisam and Disposition. 29; 435-442. 0.05 level 0.065 0.066 0.090 0.091 6. Seung I l Cho, Jiyeon Shim, Min-su kim, Yong-kweon kim, 0.01 level 0.096 0.097 0.133 0.134 Doo soo chung (2004) On-line sample clean up and chiral separation of gemifloxacin in a urinary solution using % Error in bulk samplesb 0.056 0.073 0.107 -0.055 chiral crown ether as a chiral selector in microchip a Average of eight determinations. electrophoresis. Journal of chromatography A. 1055; b Average of three determinations 241-245. In Y =a + bC, Y is absorbance and C is concentration 7. Won jae Lee, Chang Yang Hong (2000) Direct liquid chromatographic enantiomer Table 2: Results of recovery study by standard addition method separation of new fluoroquinolones Dosage Labeled % Recovery* % RSD including gemifloxacxin. Journal of -1 forms (g ml ) chromatography A. 879; 113-120. Taken+ 8. Eun sook kim, yoo-mo koo, doo soo chung. Added Chiral counter-current chromatography of Method A Method B Method C Method D Method A Method B Method C Method D gemifloxacin guided by capillary electrophoresis using (+)- (18-crown-6)Tablets-1 4+4 100.18 99.90 99.98 100.01 0.35 0.24 0.28 0.52 tetracarboxylic acid as a chiral selector. 5+5 100.10 100.32 100.60 100.20 0.41 0.21 0.53 0.36 Journal of chromatography A. 1045; 119Tablets-2 3+5 99.99 100.05 100.10 100.02 0.26 0.52 0.58 0.40 124. 4+6 100.04 100.16 100.25 100.32 0.48 0.31 0.45 0.64 Source: The Pharma Review, * Average of six independent analyses

December 2007

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Simultaneous Estimation of Aceclofenac and Paracetamol in Combined Dosage Form by Two Wavelength SpectrophotometryWadher S. J.*, Momin M. Y., Puranik M. P. and Yeole P. G.

Abstract: A simple, rapid and economical method for simultaneous estimation of aceclofenac and paracetamol in tablet formulation has been developed. Paracetamol is quantized using two wavelengths, 230 nm and 275 nm where as aceclofenac is quantized using two wavelengths, 224 nm and 269 nm in methanol. The Beer's law is obeyed by paracetamol and aceclofenac in concentration range of 0-30 g/ml and 0-50g/ml for aceclofenac and paracetamol respectively.

IntroductionAceclofenac (ACF), {[2-(2`, 6`-dichlorophenyl) amino] phenyl acetoxyacetic acid} is a new phenyl acetic acid derivative with potent analgesic and anti-inflammatory properties with improved gastric tolerance. Paracetamol (PCM), chemically 4-hydroxy acetanilide, is a centrally and peripherally acting analgesic, antipyretic agent. A combination of these two drugs containing aceclofenac (100 mg) and paracetamol (500 mg) is available commercially by different manufacturers. This combination is use for pain and management of rheumatic disorders. B.P.1 describes titrimetric method for estimation of aceclofenac. Paracetamol is official in I.P.2, B.P.1 and U.S.P.3 which describes titrimetric and spectrophotometric methods for its estimation. Only few methods4-9 have been reported for determination of aceclofenac individually, whereas many methods9-13 have been described in literature for determination of paracetamol alone or in combination with other drugs. The review of the literature revealed that no method is as yet reported for the simultaneous estimation of both the drugs in combined dosage form. Present paper described simple, accurate and reproducible two wavelength spectrophotometric method for simultaneous estimation of two drugs in tablet formulation.

used to measure absorbance of the resulting solution, A Shimadzu analytical balance model AW 220. Reagent Methanol AR grade.

Standard stock solution1. Aceclofenac standard stock solution (0.1 mg/ml) was prepared by transferring accurately weighted 10 mg of pure aceclofenac in 100 ml volumetric flask and volume was made to mark with methanol to give 10 g/ml. 2. Paracetamol standard stock solution (0.5 mg/ml) was prepared by transferring accurately weighted 50 mg of pure paracetamol in 100 ml volumetric flask and volume was made to mark with methanol to give 50 g/ml. Selection of wavelength: Selection of analytical wavelengths was done by taking pure samples of aceclofenac and paracetamol which were separately dissolved in methanol to give 20g/ml of each drug. They were scanned in the wavelength range of 200 to 400 nm. From the overlain spectra (fig.1), the wavelengths selected for paracetamol were 230 nm and 275 nm where aceclofenac has same absorbance and for aceclofenac wavelengths selected were 224 nm and 269 nm, were paracetamol has same absorbance.

Materials and MethodInstruments: A Shimadzu model 2401 double beam UV-visible spectrophotometer with a pair of 10 mm matched quartz cells was

LinearityA series of different concentration in range of 0-50 g/ml for paracetamol and 0-30 g/ml for aceclofenac were prepared from

Institute of Pharmaceutical Education and Research, Borgaon (Meghe), Wardha, M.S. *Author for correspondence Email: [email protected]

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ANALYTICAL METHOD DEVELOPMENT2.500

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Table 1: Summary of Validation Parameters Sr. No. Parameters PCM 1 Linearity Range (g/ml) Accuracy (%) Precision (R. S. D.) Ruggedness Interday (n=3) 99.12 99.89 0-30 g/ml 99.42 0.221 2 3 4

ACF 0-50 g/ml 99.52 0.235 99.70 99.50

2.000

A b s

1.500

1.000

Intraday(n=3)

0.500

Table 2: Simultaneous Assay of Aceclofenac and Paracetamol in Pharmaceutical Dosage Form Sr. No. Aceclofenac Paracetamol250.0 300.0 Wavelength (nm.) 350.0 400.0

200.0

Claim (mg) 1. 2. 100 100

Found (mg)* 99.52 100.10

Recovery (%) ** 99.88 99.71

Claim (mg) 500 500

Found Recovery (mg)* 497.90 499.20 (%)** 100.02 99.78

Fig. 1: Overlain spectra of paracetamol and aceclofenac.

stock solution. The absorbances of aceclofenac were noted at 224 nm and 269 nm and the absorbance of paracetamol were noted at 230 nm and 275 nm. The difference in absorbance of aceclofenac and paracetamol at these two wavelengths was calculated and calibration curve were plotted as absorbance difference verses concentration for aceclofenac and paracetamol. The curve shows linearity in the range of 0-35 g/ ml and 0-50g/ml for aceclofenac and paracetamol respectively.

* Average of three determination. ** After spiking the pre-analyzed sample at different levels.

Sample solutionTwenty tablets were weighed accurately, the average weight determined and then ground to a fine powder. A quantity equivalent to 100 mg of paracetamol and 20 mg of aceclofenac was weighed and transferred to a 100 ml volumetric flask. The content was shaken with methanol to dissolve the active ingredients, made to volume and filtered through Whatman filter paper no 41. The filtrate was diluted with methanol to achieve final concentration of 50 g/ml for paracetamol and 10g/ml of aceclofenac.

paracetamol in tablet formulation. The standard deviation values were satisfactory low and recovery was closed to 100 % indicating the reproducibility, accuracy and precision of proposed method. The linearity performed for both the drugs and the absorbencies were found to be linear in concentration range of 0-35 g/ ml and 050g/ml for aceclofenac and paracetamol respectively.

ConclusionThe results indicates that the proposed two wavelength method was simple, accurate and precise, hence it can be use for routine analysis which would involve determination of absorbance of sample and standard solution at the four wavelengths and simple calculation.

References1. British Pharmacopoeia, Her Majesty's Stationary office, London, UK, 2000, 32-33. 2. Indian Pharmacopoeia, Government of India, Ministry of Health and Family welfare, Vol. II, Published by the controller of publication Delhi, 1996, 484, 554-555. 3. USP 24, NF19, The United States Pharmacopoeia & National formulary, XXIV, U.S. Pharmacopoeia convention, Inc., Rockville, 2000, 17-18. 4. Hasan, N.Y., Abdel Elkawy, M. and Wagieh, N.E., Stability indicating methods for the determination of Aceclofenac, Farmaco., 2003, 58, 9199. 5. Zawilla, N.H. and Mohammad, M.A., Determination of in bulk and pharmaceutical formulations, J. Pharm. Biomed. Anal., 2002, 27, 243251. 6. Hinz, B., Auge D., Rietbrock, S. and Weren, U., Simultaneous determination of Aceclofenac in human plasma by high performance liquid chromatography, J. Biomed. Chromatogr., 2002, 17, 268-275.www.kppub.com

ResultsThe results obtained by proposed method were in good agreement with the label claim (Table 1). The additives and excipient did not in interference in analysis of the label by proposed method. The method was validated in terms of accuracy, precision, ruggedness, linearity and range. The accuracy of the proposed method was determined by recovery studies. The summery of validation parameter is shown in Table 2.

DiscussionThe present study was carried out to develop a simple, rapid, sensitive, accurate, precise and rugged spectrophotometric method for the simultaneous estimation of aceclofenac and

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ANALYTICAL METHOD DEVELOPMENT7. Sharty, Y.S., Refaat, M. and Khateeb S.Z., Stability indicating spectrophotometric and densitometric methods for determination of Aceclofenac, Drug Dev. Ind. Pharm., 2002, 28, 571 582. 8. Lee, H. S., Jeong, C. K., Choi, S. J., Kim, S. B. and Lee M. H., Simultaneous determination of Aceclofenac and Diclofenac in human plasma by narrow bore HPLC using column switching, J. Pharm Biomed. Anal., 2000, 23, 775 781. 9. Harish, L., Rau, A. R. and Arora, P., Simultaneous estimation of Paracetamol and Diclofenac sodium by HPLC, Indian Drugs, 1991, 28, 285-286. 10. Subramanian, G., Musmade, P. and Udupa, N., Simultaneous estimation of Tizanidine, Diclofenac potassium and Paracetamol in tablet, Ind. J. Pharm. Sci., 2004, 66, 694-693. 11. Patil, D., Raman, B., Simultaneous estimation of Dextropropoxyphen HCl, Diclofenac sodium & Paracetamol by RP-HPLC, Indian Drugs, 2001, 38(1), 36-39. 12. Marin, A., Garcia, A. and Barbas, C., Validation of a HPLC quantification of Acetaminophen Phenylephrine and Chlorpheniramine in pharmaceutical formulations capsules and sachets, J. Pharm, Biomed. Anal., 2002, 29, 701-714. 13. Garcia, A., Ruperez, F. J., Marin, A. and Barbas, C., Poly (ethyleneglycol) column for the determination of Acetaminophen, Phenylephrine and Chlorpheniramine in pharmaceutical formulations, J. Chromatogr., B., 2003, 785, 237-293. 14. ICH Q2A, International Conference of Harmonization: Guidelines for Industry Q2A, Text on the Validation of Analytical Procedures, Federal Register, 1996, 60, 11260-11262.

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Source: The Pharma Review, October 2007

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ANALYTICAL METHOD DEVELOPMENT

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Application of UV-Spectrophotometric Method for Estimation of Drotaverine Hydrochloride in Bulk & TabletsA. A. Shirkhedkar*, G. H. Upasani and S.J. Surana

Abstract: A simple, rapid and accurate spectrophotometric method has been developed for the quantitative estimation of Drotaverine HCl (DH) in bulk and tablets. In methanol, DH exhibits absorption at 354 nm and the method obeys Beer's law at the concentration range of 4 - 24 g/mL. The percentage label claim was found to be in range of 98-101%. The proposed method was validated statistically and by recovery studies.

IntroductionDrotaverine hydrochloride (DH), 1- (3, 4-diethoxybenzylidene)-6, 7 diethoxy 1, 2, 3, 4- tetrahydroisoquinoline is antispasmodic1 and acts by inhibiting phosphodiesterase IV enzyme, specific for smooth muscle spasm and pain, mainly in labor pain.2-3 Literature survey revealed that HPLC method for the determination of DH in human plasma and urine4 and few spectrophotometric methods5-6 are available for estimation of DH alone and in combinations with other drugs. DH is not official in pharmacopoeia. Therefore, it was thought worthwhile to develop a simple, accurate and reliable spectrophotometric method for the estimation of DH in bulk and in tablet dosage form using methanol as a solvent. All the chemicals used were of analytical grade. Spectral and absorbance measurements were made on Shimadzu UV- visible spectrophotometer- 2450 with 10mm matched quartz cells.

Table1: Optical Characteristics and Precision of The Proposed Method

Parameters Wavelength Beer's law limit (g/ml) Regression equation (Y= mx+C) Molar extinction coefficient (Lmol cm )* E 1% Slope (m) Intercept (c) Correlation coefficient (r)-1/ -1

354 nm 4 - 24 Y=0.02516x+0.0115 1.06104 230 0.0251 0.0115 0.9994

Average of six determinations*

Preparation of standard solutionAccurately weighed 10 mg of DH pure drug was dissolved in methanol to give stock solution of 100 g/mL concentration. From this stock solution, working standard solutions of drug (4-24 g/mL) were prepared by appropriate dilutions with distilled water. Working standard solutions were scanned in the UV range of (200-400nm). The optical characteristics such as Beer's law limits, molar extinction coefficient (Lmol-1/cm-1), regression equation, correlation coefficient were calculated and results are presented in Table 1.

powder. An accurately weighed powder equivalent to 10 mg of DH was transferred in to 100 mL volumetric flask and volume make up to the mark with methanol. From this stock solution, working sample solutions of drug were prepared by appropriate dilutions with distilled water. The results of assay are presented in Table 2. Table 2: Results of AssayLabel claim mg/tab 40 *Amount found mg/tab 39.9 %Label claim 99.7 % R.S.D. 1.27

Average of three determinations*

Recovery studiesAccuracy of the method was checked by recovery studies. The recovery studies were carried at 80%, 100% and 120% level. The results of recovery studies are presented in Table 3.

Preparation of sample solutionTwenty tablets were accurately weighed and ground to fine

Results and DiscussionIn methanol, DH exhibits absorption at 354 nm. The linearity was observed in concentration range of 4-24 g/mL. The amount of

R.C. Patel College of Pharmacy, Shirpur Dist: Dhule (M.S.) *Author for Correspondence E-mail: [email protected]

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ANALYTICAL METHOD DEVELOPMENTTable 3: Results of Recovery StudiesConcentration Added (mcg/mL) 8 9.9 101 *Amount recovered (mcg/mL) 10 12.1 1.3 % Recovered % R.S.D 7.9 99.8 1.7

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12 99.6 1.5

Average of three determinations at each level*

DH estimated by the proposed method was in good agreement with the label claim. The % recovery was found to be in the range of 99-101%. The low % RSD value indicates that method is accurate. The proposed method is simple, accurate, economical and be used for routine analysis of DH from tablet formulations.

References1. 2. 3. 4. 5. 6. Martindale, The Complete Drug Reference, 33rd edn. Sean C. Sweetman, The Pharmaceutical Press, London, 1606. The Merck Index, 13th edn., Merck & Co., Inc., Whitehouse station, 2004, 609-610. Singh K.C., Jain P., Goel N. and Saxena A., Inter. J. Gayneco. & Obst. 2004, 84 (1), 17-21. Bolaji O.O., Onyeji C.O., J.Chromatogr.B. Biomed. App., 1993, 622 (1), 93-97. Mahajan V.K., Dahivelkar P.P., Fursule R.A., Shirkhedkar A.A and Surana S.J., Indian Drug, 2006, 43 (8), 656-659. Dahivelkar P.P., Mahajan V.K., Bari S.B., Shirkhedkar A.A., and Surana S.J., Indian Drugs, 2006, 43 (11), 896-900.

Source: The Pharma Review, August 2007

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Oxidative Coupling, Complex Formation and Internal Salt Formation Reactions for Visible Spectrophotometric Determination of Alfuzosin Hydrochloride in Pharmaceutical FormulationsM. Vamsi Krishna*, D. Gowri Sankar

Abstract: Three simple and sensitive visible spectrophotometric methods have been developed for the determination of Alfuzosin hydrochloride (AFZ) in pharmaceutical formulations. Method A is based on the oxidative coupling reaction of AFZ with thymol under alkaline conditions in presence of sodium hypochlorite and the colored product formed was measured at 510 nm. Method B is based on the reaction of AFZ with cobalt thocyanate and the colored complex was measured at 630 nm. Method C is based on the formation of a colored internal salt between AFZ and citric acid in presence of acetic anhydride, which absorbs maximally at 580 nm. Beer's law is obeyed in the concentration ranges 8-40, 20-100 and 5-25 g/ml for method A, B and C respectively. The methods have been applied to the determination of AFZ in commercial tablets. Results of analysis were validated statistically and are found to accurate and precise.

IntroductionAlfuzosin Hydrochloride (AFZ) 1 is a alpha 1- receptor blocker and is chemically known as N-[3-[(4-amino-6, 7-dimethoxy quinazolin-2-yl)-methyl-amino]propyl]oxolane-2-carboxamide hydrochloride. Figure 1 shows the structure of alfuzosin. It is used for the treatment of lower urinary tract symptoms associated with benign prostatic hyperplasia. Literature survey reveals that, few chromatographic2-6 methods have been reported for the estimation of AFZ. To the best of our knowledge, there is no work in the literature reported about the spectrophotometric method for the analysis of AFZ in either biological fluids or pharmaceutical formulations. Hence the author has made an attempt to develop three simple and sensitive spectrophotometric methods for theNH2

estimation of AFZ in bulk drugs and in pharmaceutical formulations. Method A is based on the oxidative coupling reaction of AFZ with thymol under alkaline conditions in presence of sodium hypochlorite and the colored product formed was measured at 510 nm. Method B is based on the reaction of AFZ with cobalt thocyanate and the colored complex was measured at 630 nm. Method C is based on the formation of a colored internal salt between AFZ and citric acid in presence of acetic anhydride, which absorbs maximally at 580 nm.

ExperimentalApparatus: All spectral and absorbance measurements were made on a systronic model 117 digital spectrophotometer with 10mm matched quartz cells. Materials and reagents: All chemicals used were of analytical reagent grade and double distilled water was used for preparing the reagent solutions. AFZ was obtained from Dr. Reddy's labs Hyderabad. Stock solution of AFZ was freshly prepared by dissolving 100mg of AFZ in 100ml of distilled water and then this was further diluted with distilled water so as to obtain working standard solution of 200, 400 and 250g/ml for methods A, B and C

CH3ON O O

CH O 3

N

N

NH

CH 3

Figure 1: Structure of Alfuzosin

Pharmaceutical Analysis and Quality Assurance Division, University College of Pharmaceutical Sciences, Andhra University, Visakhapatnam. *Author for Correspondence: E-mail: [email protected]

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ANALYTICAL METHOD DEVELOPMENTrespectively. H2SO4 (50%), Aqueous NaOCl, 5% Thymol (5 g in 100 ml of ethanol), NaOH solution (40%), Cobalt thiocyanate solution (2.50x10 -1M), Buffer solution (pH 2.0), nitrobenzene and 12% citric acid in acetic anhydride were used.

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General procedureMethod A: Aliquots of working standard solution (0.4-2.0 ml, 200 g/ml) were placed separately in a series of 10 ml graduated test tubes. To that 0.4 ml of H2SO4 , 1.0 ml sodium hypochlorite solution and 0.5 ml of thymol solution were added and kept a side for 5 min at room temperature with occasional shaking. Then 1.2 ml of NaOH solution was added and all the tubes were put in water bath at 20-25 0C for 5 min and made up to 10 ml with distilled water. The absorbances were measured at 510 nm against the reagent blank. The calibration curve was constructed by plotting the absorbance versus final concentration of the alfuzosin. The content of the unknown was computed either from calibration curve or regression equation.

Assay of pharmaceutical tablets: Twenty tablets were powdered and mixed thoroughly. An amount equivalent to 100 mg of the drug was dissolved in water and filtered. The filtrate was made up to 100 ml and appropriate aliquots of the drug solution were treated as described above for the determination of AFZ.

Results and discussionAnalytical data: The optical characteristics such as Beer's law limits, Sandell's sensitivity, molar absorptivity, percent relative standard deviation (calculated from eight replicate samples containing 3/4th of the amount of the upper beer's law limits) were calculated for all the methods and the results are summarized in table 1. Regression characteristics like standard deviation of slope (Sb ), standard deviation of intercept (Sa ), standard error of estimation (Se ), % range of error (0.05 and 0.01 confidence limits) and detection limit were calculated for all the methods and are shown in table 1.

Method B: Aliquots of working standard solution (0.5-2.5 ml, Table 1. Optical and Regression Characteristics, Precision and Accuracy of the 400 g/ml) were delivered in to a series of calibrated tubes. 2 Proposed Methods for AFZ Parameter Method A Method B Method C ml of buffer of pH 2.0 and 5 ml of cobalt thocyanate solutions gmax (nm) 510 630 580 were added and the total volume in each tube was adjusted to 15 ml with distilled water. These solutions in the tubes were Beer's law limits (g ml-1) 8.0 - 40.0 20.0 - 100.0 5.0 - 25 transferred to 125 ml separating funnel. To each separating -1 Detection limits (g ml ) 0.190 0.771 0.143 funnel 10.0 ml of nitrobenzene was added and the contents -1 -1 3 3 Molar absorptivity (L mole cm ) 9.1 x 10 3.83 x 10 1.50 x 104 were shaken for 2 min. The two phases were allowed to Sandell's sensitivity 0.046 0.110 0.028 separate and the absorbance of the separated nitrobenzene (g cm-2 / 0.001 absorbance unit) layer was measured after 20 min at 630 nm against the Regression equation (Y = a + bC) reagent blank. The calibration curve was constructed by Slope (b) 2.15 x 10-2 9.06 x 10-3 3.56 x 10-2 plotting the absorbance versus final concentration of the Standard deviation of slope (Sb) 0.05 x 10-3 0.04 x 10-3 0.10x 10-3 alfuzosin. The content of the unknown was computed either Intercept (a) -0.70 x 10-3 1.0 x 10-3 -1.70 x 10-3 from calibration curve or regression equation.a Method C: Aliquots of working standard solution (0.5-2.5 ml, Standard error of estimation (Se) 1.82 x 10-3 2.22 x 10-3 250g/ml) were transferred in to a series of 25 ml graduated tubes and gently evaporated on a boiling water bath to Correlation coefficient (r) 0.9999 0.9999 a dryness. 10 ml of citric acid reagent was added to each tube. Relative standard deviation (%) 0.121 0.142 the tubes were placed in a boiling water bath and heated for a % Range of error(Confidence limits) 30 min. the solution in each tube was made up to the mark 0.05 level 0.102 0.120 with acetic anhydride, the absorbance of the colored 0.01 level 0.150 0.177 solutions was measured at 580 nm against the reagent blank. b % Error in bulk samples 0.164 -0.173 The calibration curve was constructed by plotting the a absorbance versus final concentration of the alfuzosin. The Average of eight determinations b content of the unknown was computed either from calibration Average of three determinations In Y =a + bC, Y is absorbance and C is concentration curve or regression equation.

Standard deviation of intercept (S ) 1.37 x 10-3 2.33 x 10-3

1.70 x 10-3 1.62 x 10-3 0.9999 0.130

0.109 0.161 0.211

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ANALYTICAL METHOD DEVELOPMENTAnalysis of pharmaceutical preparations: Application of the proposed methods to the determination of AFZ in its dosage forms was successfully made; the results are presented in table 2. The excellent recoveries obtained indicated the absence of any interference from the excipients.Table 2. Results of analysis of tablet formulations containing AFZ Formulation Labeled Amount (mg) Tablets-1 Tablets-2 10 10 Recovery*%RSD** Method A 99.90.31 Method B Method C 4.

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References1. Elhilali M M (2006) Alfuzosin: an alpha1-receptor blocker for the treatment of lower urinary tract symptoms associated with benign prostatic hyperplasia. Expert. Opin. Pharmacother.7: 583-596. Niu C Q, Ren L M (2002) chiral separation and preparation of three new antagonists of alpha 1-adrenoceptors by chiral mobile phase HPLC. Yao Xue Xue Bao., 37: 450-453. Wiesner J L, Sutherland F C W, Van Essen G H, Hundt H K L, Swart K J, Hundt A F (2003) selective, sensitive and rapid liquid chromatography-tandem mass Spectrometry method for the determination of alfuzosin in human plasma. J. Chromatogr., B: Anal. Technol. Biomed. Life Sci. 788: 361-368. Kratulovic A M, Vende J L (1989) Improved performance of the second generation alpha 1-AGP columns: applications to the routine assay of plasma levels of alfuzosin hydrochloride. Chirality. 1: 243-245. Rouchouse A, Manoha M, Durand A, Thenot J P (1990) Direct high performance liquid chromatographic determination of the enantiomers of alfuzosin in plasma on a second generation alpha 1-acid glycoprotein chiral stationary phase. J. Chromatogr., 506: 601-610. Guinebault P, Broquaire M, Colafranceschi C, Thenot JP (1986) High Performance liquid chromatographic determination of alfuzosin in biological fluids with fluorimetric detection and large volume injection. J. Chromatogr. 353: 361-369.

2.

3.

100.10.65 100.060.55

100.10.50 100.080.38 100.20.40

* Average of 5 determinations ** Relative standard deviation

ConclusionsThe data given above reveal that the proposed methods are simple, accurate and sensitive with good precision and accuracy. The proposed methods can be used as alternative methods to the reported ones for the routine determination of alfuzosin. This encourages their successful use in routine analysis of alfuzosin in quality control laboratories

5.

6.

Source: The Pharma Review, June 2007

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ANALYTICAL METHOD DEVELOPMENT

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Development of Spectrophotometric Analysis Method of Atenolol Gel FormulationsNandy B. C, Gupta R.N

Abstract: Atenolol is a slightly water soluble drug which has about 50% bioavailability through the conventional oral route. It is the most commonly used selective b1 blocker in hypertension treatment. Generally, so many techniques are used to increase the aqueous solubilities of slightly water soluble drugs. To extract the drug candidates from the gel formulations organic solvents are used mostly as co-solvents. In this proposed method methanol used as a co-solvent with the phosphate buffer (pH 7.4) to extract the drug from the gel formulations. Atenolol shows maximum absorbance at near about 224 nm. Results of analysis were validated statistically and by the stability and recovery studies. The proposed investigation is new, simple, accurate, reproducible and also cost effective. Thus it can be successfully employed in routine analysis of atenolol gel formulations which can be applied for novel iontophoretic delivery system.

IntroductionAtenolol is a selective b1-adrenergic blocking agent used in the treatment of various cardiovascular disorders. With an oral bioavailability of 50%, the transdermal delivery of these drugs could be a potential alternative to oral delivery to increase therapeutic efficacy, bypassing hepatic first-pass metabolism and low oral absorption.1 Literature survey reveals determination of Atenolol by Spectrophotometric, HPLC, GC and Spectroflurimeteric methods. Although, most of the analytical methods of Atenolol formulations, they emphasized on HPLC estimation procedures. An attempt has been made in this proposed work to develop simple, cost effective and accurate UV and visible spectrophotometric methods for the estimation of drug from the gel formulations. The reported lmax for Atenolol drug in methanol at 224nm. So, many journals they used only methanol as a co-solvent to extract the drug contents from the various pharmaceutical formulations and also used simple spectrophotometric method, likes Norfloxacin,5 Clozapine,6 Citalopram Hydrobromide7 in tablets dosage form and Piroxicam,8 sodium nonivamide acetate II,9 b blockers10 & Atenolol11-13 in gel formulation.2-4

Chemicals: The drug Atenolol was received as gift sample from Stadmed Pvt. Ltd., Kolkata and others reagents used all were analytical grades.

Scanning of Atenolol solution in various solvents10mg of Atenolol was dissolved in 100ml of respective solvents phosphate buffer (pH7.4) and phosphate buffer (pH7.4) with 5% methanol solution), so as a solution of 100g/ml was prepared as a stock solution. From his 10ml was taken and the volume was made up to 50 ml to make solution concentration 20g/ml. The resulting solution was scanned by using spectrophotometer (Model Shimadzu, UV-Pharma spec 1700: UV-Visible Spectrophotophotometer). Scanning is reported in Fig. no. 1a and 2a respectively.

Preparation of standard plot of Atenolol in different solutions10mg of Atenolol was dissolved in 100ml of different solution phosphate buffer (pH7.4) and phosphate buffer (pH7.4) with 5% methanol solution, so as a solution of 100g/ml was prepared as a stock solution. From his 2.5,5,7.5,10,12.5,&15ml was taken and the volume was made up to 50 ml to make solution concentration of 5,10,15,20,25,30g/ml respectively and absorbance were measured triplicate at wavelength maxima 224.2nm, 224.2 and 224.3 respectively. All standard curves are mentioned in Fig. no.1b and 2b in respective solutions.

Materials and MethodsInstrument: Shimadzu, UV-Pharma spec 1700: UV-Visible Spectrophotophotometer.

Department of Pharmaceutical Science, Birla Institute of Technology, Mesra, Ranchiwww.kppub.com

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ANALYTICAL METHOD DEVELOPMENTTable 1: lmax values of atenolol solution in different solvents (concentration 20 g/ml)S.No. Solvent 1 20.693 0.600

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l max in nm 224.2 224.3

(pH7.4) by heating on a water bath for 30 min to effect complete solution & keep it to cool at the room temperature & stirred that preparation using mixer until a clear gel were obtained. Methyl paraben and propyl paraben solution were prepared by heating with the some portion of propylene glycol until a clear solution were obtained. Then this solution was mixed with the gel preparation. Atenolol 1.5% w/w dispersed into the propylene glycol and l-menthol 2%w/w dissolved into the propylene glycol but in case of tween-20 incorporated to the polymer solutions directly and dissolved by continuous stirring at about 500 rpm for 30 min. the resulting solution was then adjusted to ph 7.4 by the addition of the 1M NaOH and/ or 1 M HCl solution. The solution was then stored at room temperature to ensure complete polymer dissolution in an air tight glass container.

Phosphate buffer ( pH7.4 ) Phosphate buffer ( pH7.4 ) with 5 % methanol1.033403

Abs.

Abs.2

0.400

0.50000

0.2001

0.00000

0.000 210.000 220.000

240.000

260.000 nm.

280.000

300.000

0.00000

10.00000

20.00000 Conc. (mcg/ml)

30.00000

y=0.03113x+0.00566 r2 Correlation Coefficient = 0.99796

Fig. 1: (a) lmax values of atenolol of 20g/ml solution in Phosphate buffer solution (pH 7.4) was found at 224.2 and (b) its standard curve.0.706 0.600 1.04772 1.000003

Abs.

0.400Abs.

0.50000

Recovery studiesIn order to check the accuracy, precision and the reproducibility of the proposed method, recovery studies were conducted.

0.2002 1

0.000 210.000 220.000

240.000

260.000 nm.

280.000

300.000

0.00000 -0.097471 0.00000

10.00000

y=0.03113 x + 0.00965 r2 Correlation Coefficient = 0.99951

20.00000 Conc. (mcg/ml)

30.00000

Fig. 2: (a) lmax values of atenolol of 20g/ml solution in Phosphate buffer solution (pH 7.4) with 5% methanol was found at 224.3 and (b) its standard curve.

Solution StabilityTo ensure the stability of the drug during the analysis as well as stability of drug in various formulations, a short term solution stability study was carried out.

The drug solution of strength 20g/ml and 30g/ml were prepared with the phosphate buffer (pH 7.4) and solutions pH were adjusted to 7.4 with the 1M HCL and/ or 1 M sodium hydroxide solutions as per required and the solutions were Table 2: Stability of the drug in phosphate buffer solution (pH7.4) kept at 37C for 48 hours and the samples withdrawn at 0, 24 Theoretical 20g/ml 30g/ml a n d 4 8 h o u r s . T h e s a m p l e s w e r e a n a l y z e d conc. of drug solution spectrophotometrically at 224.2nm

After accurately weighing 1 gm of gel preparation of different formulations (Table no.3), which contains equivalent to 15 mg of Atenolol was transferred to a 100ml volumetric flask. Now about 50 ml of respective solvent Phosphate buffer (pH 7.4) and another was phosphate buffer with 5% methanol were added separately and flask were shaken for about 15 min to solubilize the drug. Then the volumes were made up to the mark with respective solvents and which were used as stock solutions.

0 24 48 0 24 48 The tabulated results indicate that there was no significant Time interval (hrs) decrease in the absorbance after 48 hours. Hence the drug Absorbance at g 0.6267 0.6247 0.621 0.9407 0.9403 0.9383 was found to be stable in the media and also the % drug max 224.2 (n=3) 0.00125 0.00094 0.00081 0.00125 0.00125 0.00125 contents all are within the limit.

Procedure

% drug content

99.75 99.43 98.83 100.12 100.07 99.87 0.20 0.14 0.13 0.14 0.14 0.12 0.20 0.14 0.131 0.139 0.14 0.12

Sodium carboxy methyl cellulose based gel was prepared by % Coefficient Of dissolving polymer as required amount of phosphate buffer variation

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ANALYTICAL METHOD DEVELOPMENTPreparation of gel formulationsTable 3: Formula used in different gel preparations of Atenolol.S.N. Materials F1, F2 &F3 F4 ,F5 &F6 %(w/w) %(w/w) F7%(w/w) F8%(w/w) Blank Blank

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Table 5: Recovery studies by using phosphate buffer solution (pH7.4) for Atenolol gel with l-menthol as a penetration enhancer and its statistical evaluation. Gel Equivalent Dilution of formulations amount of Stock solution Atenolol in gel (mg) F4 F5 F6 15 15 15 15 15 15 %Recovery of Atenolol (Mean S.D.)

1. 2. 3. 4. 5. 6. 7.

Atenolol Sodium CMC Methyl paraben Propyl paraben Propylene glycol L-Menthol Tween- 20

1.5 3.0 0.2 0.02 30 5

1.5 3.0 0.2 0.02 30 2 -

3.0 0.2 0.02 30 5

3.0 0.2 0.02 30 2 -

1ml up to 10ml 98.8690.253 2ml up to 10ml 99.6540.129 1ml up to 10ml 99.3690.251 2ml up to 10ml 99.6900.100 1ml up to 10ml 98.4400.250 2ml up to 10ml 99.5830.136

Then the solutions were filtered through Whatmann filter paper 41. The 1ml and 2ml filtrates of these each solutions were transferred to two different, 10 ml volumetric flasks separately and volume made up to 10 ml with the respective solvents. Blank preparations were also carried out by the same method of two different formulations containing respective penetration enhancers, which having no drug contents. Then the absorbance were noted at 224.2nm for phosphate buffer (pH 7.4) solution and 224.3 nm for phosphate buffer (pH 7.4) with 5% methanol solution, against corresponding blank preparation. Drug content and % recovery were calculated. (Table 4, 5,6 and 7).Table 4: Recovery studies by using phosphate buffer solution (pH7.4) for Atenolol gel with tween-20 as a penetration enhancer and its statistical evaluation. Gel Equivalent Dilution of formulations amount of Stock solution Atenolol in gel (mg) F1 F2 F3 15 15 15 15 15 15 %Recovery of Atenolol (Mean S.D.) % Coeff. Of variation 0.100 0.015 0.174 0.087 0.031 0.100

Table 6: Recovery studies by using phosphate buffer solution (pH7.4) with 5 % methanol for Atenolol gel with tween-20 as a penetration enhancer and its statistical evaluation. Gel Equivalent Dilution of formulations amount of Stock solution Atenolol in gel (mg) F1 F2 F3 15 15 15 15 15 15 %Recovery of Atenolol (Mean S.D.) % Coeff. Of variation 0.333 0.263 1.050 0.260

1ml up to 10ml 100.360 0.170 0.169 2ml up to 10ml 100.1820.334 1ml up to 10ml 100.2980.264 2ml up to 10ml 100.860 1.06 1ml up to 10ml 99.870 0.260

2ml up to 10ml 101.200 0.190 0.187

Table 7: Recovery studies by using phosphate buffer solution (pH7.4) with 5 % methanol for Atenolol gel with l-menthol as a penetration enhancer and its statistical evaluation. Gel Equivalent Dilution of %Recovery % formulations amount of Stock solution of Atenolol Coeff. Atenolol in (Mean S.D.) Of gel (mg) variation F4 F5 F6 15 15 15 15 15 15 1ml up to 10ml 99.7940.185 2ml up to 10ml 100.1820.061 1ml up to 10ml 100.2200.29 2ml up to 10ml 100.3200.26 1ml up to 10ml 99.5100.17 2ml up to 10ml 100.2200.22 0.185 0.060 0.289 0.259 0.170 0.219

1ml up to 10ml 99.3770.100 2ml up to 10ml 99.9700.015 1ml up to 10ml 99.4400.174 2ml up to 10ml 99.8320.087 1ml up to 10ml 99.8050.031 2ml up to 10ml 99.8650.100

Result and DiscussionThe solubility of Atenolol in Phosphate buffer (pH 7.4) with 5% methanol solution was found to be higher as compared to its solubility in distilled water and phosphate buffer solution. It has also been seen that the solubility of Atenolol in distilled water and phosphate buffer is almost same. Thus it is concluded that the enhancement of solubility is due to the addition of the methanol which act as a co-solvent in the solution. Also it has been already

reported that Atenolol is freely soluble in methanol and slightly soluble in water. Hence, from the stability study of the atenolol in PB solution (pH 7.4), as per evident from the table 2), the % drug content after 48 hrs aged solution (kept at 37c temperature) of drug in PB (pH7.4) solutions were fount to have same and near bywww.kppub.com

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ANALYTICAL METHOD DEVELOPMENT100% drug contains with out any degradation at pH 7.4 and also% coefficient of variation and standard deviation are very low. So the proposed method is accurate and which is statistically validated. This indicates that the analysis can be performed accurately with in 48 hrs of extraction of drug from the gel formulations. The results of recovery studies (table 4 and 5), which indicates that the % recovery by using PB (pH 7.4) gives the estimated, ranged from 99.377 0.10 to 99.865 0.10, in case of gel preparation where in tween-20 used as a penetration enhancer and for l-menthol 99.3690.251 to 99.869 0.2508. But the % recovery is almost 100% and above when used PB (pH7.4) with 5% methanol (table no. 6 and 7) as an extracting solvent, this indicates the accuracy of the proposed method. A value of standard deviation, % coefficient of variation are satisfactorily low and confirms further reproducibility, uniformity, consistent and precision of the proposed method. Although so many journals are used to determine the gel formulations via HPLC method, but widely used spectrophotometeric estimations of slightly water soluble drugs can be useful to perform in a precise manner. Although the g max values were used here at about 224.2nm and 224.3 nm, which are not affected by the water and methanol because the cut off wave lengths of that solvent are 180nm and 210 nm respectively. Since there are also other two peaks at around 275nm & 283nm, but these are having lower absorbance difference with increasing the concentration concurrently.

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References1. Denet, A. R.; Ucakar, B.; Preat, V. Transdermal delivery of Timolol and Atenolol using electroporation and iontophoresis in combination: A mechanistic approach. Pharm. Res. 2003, 20, 1946-1951. Caplar, V.; Mihun, Z.M.; Hofman, H.; Kuftinee, J.; Kajfez, F.; Nagl, A.; Blazevic, N. Atenolol. In Analytical profiles of drugs substances. Florey, K. Ed. Acadmic press: New Yark, 1984; 13, pp 1-25. The Indian pharmacopoeia, Vol.1, Controller of publications: Delhi, 1996; pp 72-74. Dollery, C. Therapeutic Drugs. Ed. Churchill livirgstone: London, 1999; 1, PP A224-A227. Maheshwari R.K.Application of hydrotropic solubilization phenomenon in Spectrophotometeric estimation of Norfloxacin in tablets. Ind. J. Pharm. Edu. Res. 2006, 40, 237-240. Kuchekar B.S.; Gavhane Y. N.; Gaikwad N. B.; Thakkar S. V. Spectrophotometric estimation of Clozapine in tablets. Ind. J. Pharm. Edu. Res. 2006, 40,203-204. Pillai, S.; Singhvi I. Spectrophotometric methods for quantitative estimation of Citalopram Hydrobromide from tablet formulation. Ind. J. Pharm. Edu. Res. 2006, 40, 175-177. Attia , M. A., Gibaly, I. E., Shaltout, S.E., Fetih, G.N. 2004. Transbuccal permeation, anti-inflammatory activity and clinical efficacy of piroxicam formulated in different gels. Int. J. Pharm.276, 11-28. Fang, J.Y.; Huang, Y.B.; Wu, P.C.; Tsai, Y.H. 1996. Transdermal iontophoresis of sodium nonivamide acetate II: optimization and evaluation on solutions and gels. Int. J. Pharm.145, 175-186.

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10. Salem H. Spectrophotometric determination of beta-adrenergic blocking agents in Pharmaceutical formulations. J. Pharm. Biomed. Anal. 2002, 29, 527-538. 11. Bhaskaran, S., Harsha, N. S. 2000. Effect of permeation enhancer and iontophoresis on permeation of atenolol from transdermal gels. Ind. J. Pharm. Res. 6, 424-426. 12. Al-Ghannam S.M.; Belal F. Kinetic Spectrophotomtric determination of atenolol in dosage forms. J AOAC Int. 2002, 85, 817-823. 13. Vetuschi C.; Ragno G. Fourth UV derivative spectrophotometry for the simultaneous assay of atenolol and chlorthahlidone in pharmaceuticals. Int. J. Pharm. 1990, 177-181.

ConclusionIt is thus concluded that the proposed method is new, simple, and also accurate, cost effective, and precise and not so time consuming. The proposed method can be successfully employed in the routing analysis of Atenolol in gel formulations which are generally used for iontophoresis delivery system as effective novel transdermal preparations.

Source: The Pharma Review, June 2007

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UV-Spectrophotometric Estimation of Nabumetone$M. N. Purohit *, Sharad Mohan, Kunal Chokshi, G. V. Pujar

Abstract: A simple and sensitive spectrophotometric method has been developed for determination of nabumetone in bulk powder and its tablet dosage form. The method is found to be linear over the concentration range of 10-35 g/ml. for method A and 8-25 g/ml. for meth