Available online at www.pharmscidirect.com Int J Pharm Biomed Res 2012, 3(2), 90-93 International Journal of PHARMACEUTICAL AND BIOMEDICAL RESEARCH ISSN No: 0976-0350 Research article Development and validation of spectrophotometric methods for simultaneous estimation of norfloxacin and tinidazole in combined dosage form A.J. Shinde*, H.N. More, S.R. Jadhav, V.S. Ingole, H.R. Kakade Department of Pharmaceutics, Bharati Vidyapeeth College of Pharmacy, Kolhapur-416 013, Maharashtra, India Received: 11 May 2012 / Revised: 24 May 2012 / Accepted: 26 May 2012 / Online publication: 01 Jun 2012 ABSTRACT The present work relates with two simple, accurate and reproducible spectrophotometric methods have been developed for the simultaneous estimation of Norfloxacin and Tinidazole in pharmaceutical dosage forms. The first method involves determination using the AUC (Area under Curve) method; the sampling wavelengths selected are 272-282nm and 313323nm over the concentration ranges of 2-12μg/mL and 3-18μg/mL for Norfloxacin and Tinidazole, respectively. The second method involves determination using the Q-analysis method (Absorbance Ratio Method); the sampling wavelengths selected are 277nm and 318nm over the concentration ranges of 2-12μg/mL and 3-18μg/mL for Norfloxacin and Tinidazole, respectively. The % recovery was between 98-102% indicating high degree of accuracy of the proposed methods. The results of the analysis were validated statistically and recovery studies were carried out as per ICH guidelines. The developed methods are simple, rapid, precise, accurate and can be employed for the routine estimation of NF and TZ in both bulk and tablet dosage form. Key words: Norfloxacin, Tinidazole, AUC method, Q-Analysis method 1. INTRODUCTION Norfloxacin (NF), [1-ethyl-6-fluoro-1, 4-dihydro-4-oxo7-(piperazin-1-yl) quinoline-3 carboxylic acid], is a fluoroquinolone carboxylic acid derivative used as broadspectrum antibacterial (Fig.1). The mechanism of action of Norfloxacin is through the inhibition of bacterial gyrase, an enzyme involved in DNA replication, recombination and repair. By interfering with gyrase, Norfloxacin arrest bacterial cell growth. The affinity of quinolones to metal ions seems to be an important prerequisite of their antibacterial activity: probably, quinolones bind to the DNA-gyrasecomplex via a magnesium ion . NF is the subject of a monograph in each of British Pharmacopoeia, (BP)  and the United States Pharmacopoeia, USP . The BP and USP recommended non aqueous titration for the raw material and HPLC (High Performance Liquid Chromatography) methods *Corresponding Author. Tel: +91 9823866196 Fax: +91 231 2638833 Email: [email protected] ©2012 PharmSciDirect Publications. All rights reserved. for tablets. Because of the therapeutic importance of NF, numerous analytical methods have been developed for its determination. In bulk, pharmaceutical formulations and/or biological fluids Spectrophotometric technique is the most widely used in pharmaceutical analysis [4-7]. Literature survey revealed that a number of methods have been reported for estimation of Norfloxacin  and Tinidazole [9-10] individually or in combination with other drugs. Other analytical methods have been used such as, HPLC [11-13], electrochemical analysis [14-15], Difference spectroscopy , and capillary electrophoresis . Tinidazole (TZ), [1-(2-(ethylsulfonyl) ethyl)-2-methyl-5nitroimidazole], is an effective antiprotozoal and antibacterial agent (Fig. 1). Tinidazole appears to be a promising agent for the treatment of trichomoniasis, giardiasis, amebiasis, and amebic liver abscess. TZ is the subject of monograph in each of the BP and the USP. The BP and USP recommended non aqueous titration for determination of TZ. There are several reports on the determination of TZ, both in formulations and biological fluids, viz: spectrophotometry [18-19], HPLC , and titrimetric and spectrophotomertic analysis , A.JJ. Shinde et al., Int In J Pharm Biomed Res 2012, 3(2), 90-93 91 disssolved in aceetate buffer pH H 4 and soniccated for 10miin and vo olume made to t 100mL w with acetate buffer. b It wass then filttered throughh Whatmann ffilter paper No.41. N The soolution waas suitably diluted with aacetate bufferr to obtain saample solutions contaiining NF and TZ in the con ncentrations raatio of 2:3 3, respectiveely as in the formullation. The final concentrations are a 4μg/mL off NF and 6μg//mL of TZ. Fig.1. Structural formulae of a) Norfloxacin N (NF) and b) Tinidazolee (TZ) 2.5 5 Method A (A AUC method) pootentiometry . The Norfloxacin N a and Tinidazoole cofoormulation has as an exxtended rangge of antimiccrobial sppectrum and is effective inn mixed gut infections, diiarrhea annd dysentery.. Both drugs were simultaaneously deterrmined byy spectrophootometry, HP PLC, electroochemical annalysis, caapillary electtrophoresis annd differencee spectrophotoometry sttability indicatting assay meethod . In the pressent work, AUC A Method (Area under Curve M Method) and Q-analysis Q meethod (Absorbbance Ratio Method) M sppectrophotom metric methodd is describedd for simultaaneous deetermination of o NF and TZ Z in the presennce of each other o in puure form and in pharmaceuutical dosage forms. f For the AUC C Method, 2772-282nm, and 313-323nm m were sellected as the two samplingg wavelength hs. Fig.2 repreesents thee overlain UV V spectra of N NF and TZ. NF F and TZ exhhibited lin nearity with abbsorbances inn the range off 2-12μg/mL and a 318 8μg/mL at thheir respective selected wavelengths. For sim multaneous estimation of NF and TZ Z, mixed stanndards containing NF and a TZ in a cconcentration ratio of 2:3μ μg/mL each were preppared by apprropriate dilutiion of the staandard sto ock solutions with acetatee buffer pH4.. The areas of o the miixed standardd solutions w were measureed at the selected waavelengths. A set of twoo simultaneou us equations were esttablished usinng the mean absorptivity coefficients of o NF an nd TZ at the seelected waveleength intervals. 2.. MATERIAL LS AND METHODS 2..1 Instrumentaation A Jasco UV V/Visible specctrophotometeer, model V 6330 was em mployed withh spectral banndwidth of 2nnm and waveelength acccuracy of ± 0.5nm, with automatic waavelength corrrection w employedd. A Shimadzzu electronic analytical balance was b (A AX-200) was used for weiighing the sam mple. An ultrrasonic baath sonicator was w used for sonication s of the t sample solution. 2..2 Reagents and Chemicalss Analytical pure sampless of NF and TZ were obbtained frrom Hindustann Antibiotic Limited, L Pimppri, Pune, Indiia. The gllacial acetic acid a was obtaiined from Purre-Chem Lab.. Pune. Sodium hydroxxide pellets obtained o from m Universal laab. The phharmaceuticall dosage form m used in this study s was Hinndustan A Antibiotic Lim mited, Pimpri, Pune, India labelled to contain c 4000mg NF andd 600mg of TZ Z. 2..3 Preparationn of standard stock solutionn Standard sttock solutions (100μg/mL) of NF and TZ Z were prrepared by diissolving sepaarately 10 mg m of drug each e in 1000mL acetatee buffer pH 4. The workingg standard solutions off these drugss were obtainned by dilutioon of the resppective sttock solution with w acetate buffer. b Fig.2. Overlain O spectra of NF and TZ in AUC A method A1 1 = 1.52 CNF + 0.1063 CTZ Z ---------- Eq q.(i) at 272-2882nm (λ1- λ2) A2 2 = 0.44 CNF + 0.40 CTZ ---------- Eq q.(ii) at 313-3223nm (λ3 3- λ4) Where, W 1.52 and 0.44 are meean absorbtiviity values of NF at (λ1-λ2) and (λ3--λ4), respectivvely. 0.1 1063 and 0.400 are mean abbsorbtivity vaalues of TZ at a (λ1λ2 2) and (λ3- λ44), respectivelyy. A1 1 and A2 are the t absorbancce of mixed sttandards at (λ1- λ2) an nd (λ3- λ4), resspectively. CN NF and CTZ are a concentrattions of NF an nd TZ. Th he concentratiion of CNF aand CTZ in mixed m standarrd and tab blet formulatiion can be obbtained by so olving equatioons (i) an nd (ii). 2..4 Preparationn of sample sttock solutions 2.6 6 Method B (A Absorbance raatio or Q-anallysis method) An accuratelly weighed poowder sample equivalent too 40mg off NF was trransferred to a 100L voolumetric flassk and From the overlain speectrum of NF N and TZ,, two waavelengths weere selected; one at 277nm m λmax of NF F and A.JJ. Shinde et al., Int In J Pharm Biomed Res 2012, 3(2), 90-93 otther is 310nm m, isoabsorpttive point forr both the drrugs as shhowed in Figg.3. The standdard and sam mple solutionss were prrepared in thee same manneer as in the previous methood and abbsorbance measured m at 277nm 2 and 318nm. 3 The drugs shhowed lineariity in the conncentration rannges of 2-12μ μg/mL, 3--18μg/mL forr NF and TZ respectively. r S mixed staandards Six inn ratio of 2:33μg/mL show wing linearityy within the Beer’s cooncentration range of NF N and TZ were preparred by apppropriate dillution of standdard stock sollutions (100μg/mL). T method em The mploys Q valuues; the concenntrations of drrugs in saample solutioon were deteermined by using u the folllowing eqquations. CNF = × ----------- Eq.(iii) CTZ = × ----------- Eq.(iv) 92 concentrations of each druug in samplle solutions were M calculated usingg equations (ii) and (ii) forr the AUC Method nd equations (iiii) and (iv) foor the Q-analysis method. an 2.8 8 Validation The proposeed methods w were validated d according too ICH Q2B guideliness for validation of analyttical procedurres in ord der to determ mine the linearity, sensitiv vity, precisionn and accuracy for thee analyte .. 2.8 8.1 Accuracy To ascertainn the accuraacy of the proposed p metthods, reccovery studiees were carrried out by standard adddition meethod at three different leveels (80%, 100% % and 120%). 2.8 8.2 Linearity w where, Q0 = Q1 = Q2 = A A A F A F A Z A Z A = Absorbance of sample at isoabsorpttive point; a1 and a2 abbsorptivities of o NF and TZ Z, respectivelly at isoabsorrptivity pooint. The linearityy measuremeent was evalu uated by anallyzing diffferent concenntration of thee standard so olution of NR RF and TIIZ. For AUC method and Q-analysis, the t Beer-Lam mbert’s concentration raange was founnd to be 2-12 2μg/mL for NF N and 3-18μg/mL for TZ, T respectiveely. 2.8 8.3 Precision Precision was w studied too find out in ntra and inteer-day vaariations in thhe test methood of NF an nd TZ. Calibration cu urves preparedd in medium w were run in trip plicate in sam me day an nd for three daays. % RSD (rrelative standaard deviation)) were calculated whicch should bee less than 2%. The resuults of staatistical validaation data are given in Table 1. The resuults of thee analysis andd statistical validation dataa of recovery study off the tablet form mulation are ggiven in Tablee 2 and Table 3. Tab ble 1 Op ptical characteristiics and validationn data of NF and TZ Paarameters Norffloxacin Tinidazole Methhod A Method B Method A Metthod B 277 277 318 318 2-12 2-12 3-18 3-188 Fig.3. Overlain Spectra S of NF andd TZ in Q analysiis Method (Absorrbance Ratiio Method) 2..7 Assay of tabblet formulatiion Powder equuivalent to 100mg of NF annd 10mg of TZ T was w weighed and transferred 100mL volumetric flaskk and diissolved in 500mL acetate buffer b pH 4 and a volume iss made upp with water upto100mL u affter the ultrasoonication for 15min. T solution was The w then filtereed through Whhatmann filterr paper N No.41 and dilluted further to obtain finnal concentrattion of 4μg/mL of NF and 6μg/mL of o TZ. The saample solutionns were annalyzed as per p the proceedure for mixxed standards. The Waavelengths (nm) Lin nearity & Range (µg/mL) Preecision* Interday (%RSD D) 0.4677 0.670 0.234 0.3445 Intraday (%RSD D) 0.1355 0.105 0.458 0.2332 LO OD (µg/mL)* 2.4800 0.788 2.330 1.4990 LO OQ (µg/mL)* 7.5400 1.550 1.342 5.5882 Reegression values Slope* 1.5200 0.155 0.874 0.0445 r2* 0.99999 0.9997 0.9997 0.99998 *D Denotes average of six estimations;; Method A – AU UC method ; Methhod B – Q-aanalysis method (Absorbance ( ratioo method) 3. RESULTS AND A DISCUS SSION Under the experimental e cconditions described, calibbration cu urve, assay off tablet and reecovery studiies was perfoormed. Th he developed methods m weree validated as per ICH guiddelines forr linearity, reepeatability, iintermediate precision p (inteer-day A.J. Shinde et al., Int J Pharm Biomed Res 2012, 3(2), 90-93 Table 2 Statistical validation data of tablet formulation and results of commercial sample analysis (n=6) Component Method Label claim Amount (mg/tablet) obtained (mg/tablet) NF A 400 404.25 B 400 393.6 TZ A 600 600.86 B 600 607.31 % Amount found 101.06 98.4 100.14 101.22 SD % RSD 0.0239 0.0243 0.0181 0.0391 0.1640 0.1764 0.1168 0.2530 Table 3 Statistical validation of recovery studies (n=3) Level of % recovery Methods 80 A B A B A B 100 120 % Mean recovery NF TZ 99.54 100.24 99.18 99.82 99.72 99.28 99.59 100.39 100.11 99.81 99.63 100.21 % RSD NF 0.6248 0.4621 0.3523 0.2951 0.8429 0.3982 TZ 0.8731 0.5425 0.5847 0.5866 0.6825 0.1751 93 List of symbols and Abbreviations: % : Percent Nm : Nanometer µg/mL : Microgram per Mililiter UV : Ultraviolet HPLC : High Performance Liquid Chromatography High Performance Thin Layer HPTLC : Chromatography NF : Norfloxacin TZ : Tinidazole AUC : Area under curve ICH : International conference on Harmonization SD : Standard deviation RSD : Relative Standard deviation LOD : Limit of Detection LOQ : Limit of Quantitation REFERENCES and intra-day precision studies), LOD, LOQ as shown in Table 1. 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