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1 Stability Indicing RP-HPLC Mthod Dvlopmnt and Validion for th Simultanous Estimion of Mupirocin and Mtronidazol in Pharmacutical Dosag Form Arapda Priti * 1, Marvaniya Vanita 2, Pani Pragnsh 3 1. Studnt of M.Pharm, Quality Assuranc dpartmnt, A-on Pharmacy Collg, Ahmdabad, Gujar, India 2. Assistant Profssor, Dpartmnt of Pharmacutical Chmistry, A-on Pharmacy Collg, Ahmdabad, Gujar, India 3. Profssor, Pharmacology Dpartmnt, A-on Pharmacy Collg, Ahmdabad, Gujar, India Articl history: Rcivd 07 March 2018 Accptd 02 April 2018 Availabl onlin 10 April 2018 Cition: Arapda P., Marvaniya V., Pani P. Stability Indicing RP-HPLC Mthod Dvlopmnt and Validion for th Simultanous Estimion of Mupirocin and Mtronidazol in Pharmacutical Dosag Form J Pharm Sci Bioscintific Rs (1): *For Corrspondnc: Arapda Priti Studnt of M.Pharm, Dpartmnt of Pharmacutical Chmistry, A-on Pharmacy Collg, Ahmdabad, Gujar, India. ( ABSTRACT: A simpl, rapid, conomical, prcis and accur Stability indicing RP- HPLC mthod for simultanous stimion of Mtronidazol and Mupirocin in thir combind dosag form has bn dvlopd. A rvrs phas high prformanc liquid chromographic mthod was dvlopd for th simultanous stimion of Mtronidazol and Mupirocin. In thir combind dosag form has bn dvlopd. Th sparion was achivd by LC- 20 AT C18 (250mm x 4.6 mm x 2.6 µm) column and buffr (ph 3.5): Mthanol (70:30) as mobil phas, a flow r of 1 n. Dtction was carrid out 230 nm. Rtntion tim of Mtronidazol and Mupirocin wr found to b and min, rspctivly. Th mthod has bn validd for linarity, accuracy and prcision. Linarity obsrvd for Mtronidazol 5-15 μg/ml and for Mupirocin μg/ml. Dvlopd mthod was found to b accur, prcis and rapid. Th drug was subjctd to strss condition of hydrolysis, oxidion, photolysis and thrmal dgradion, considrabl dgradion was found in alkalin dgradion. Th proposd mthod was succssfully applid for th simultanous stimion of both th drugs in commrcial combind dosag form. KEY WORDS: Mtronidazol, Mupirocin, Stability indicing RP-HPLC Mthod, Validion. INTRODUCTION: Mupirocin (Fig.1) is chmically 9-[(E)-4-[(2S,3R,4R,5S)-3,4- dihydroxy-5-[[(2s,3s)-3-[(2s,3s)-3 hydroxybutan-2- yl]oxiran-2-yl]mthyl]oxan-2-yl]-3-mthylbut-2- noyl]oxynonanoic acid. Muciprocin is an antibiotic and usd as an antibactrial agnt topically. It is ffctiv against th gram positiv bactria. It is mainly usd to tr th skin infctions. It is an antibiotic th works by stopping th growth of crtain bactria. It works by rvrsibly binding to bactrial isolucyl-t-rna synthtas, an nzym which promots th convrsion of isolucin and trna to isolucin t-rna. Prvntion of ths nzyms from functioning proprly rsults in th inhibition of bactrial protin and RNA synthsis. 1 Mupirocin is official in Indian pharmacopoia (IP 2014) 2, Th Unitd Sts Pharmacopial Convntion (USP 2010) 3 and British pharmacopoia (BP 2009) 4. Figur 1: Mupirocin Mtronidazol (Fig.2) is chmically 2 (2 mthyl 5 nitro 1H imidazol 1 yl)thanol. 117
2 Mtronidazol is usd in th trmnt of anarobic infctions and mixd infctions, surgical prophylaxis rquiring anarobic covrag, Clostridium difficilassocid diarrha and colitis, Hlicobactr pylori infction and duodnal ulcr disas, bactrial vaginosis, Giardia lamblia gastro-ntritis, ambiasis causd by Entamoba histolytica, acn rosaca (topical trmnt), and Trichomonas infctions 5. METRONIDAZOLE is official in Indian pharmacopoia (IP 2014) 6, Th Unitd Sts Pharmacopial Convntion (USP 2010) 7 and British pharmacopoia (BP 2016) 8. Figur 2: Mtronidazol Th Combinion of formulion giv synrgistic ffct Combind Dosag form of Mupirocin and Mtronidazol usd in th Trmnt of Skin Disas. Combinion of th pharmacutical formulion is widly usd in Nonhaling ulcr, Diabtic foot ulcr, Vnous ulcr, Prssur ulcr, Surgical Wound & Othr infctd Wound. Litrur rviw rvals th various mthods ar rportd for th analysis of individual drug and in combinion with othr drugs but no stability indicing RP-HPLC mthod has not bn rportd for simultanous stimion of Mtronidazol and Mupirocin Thrfor, th goal of th prsnt work is to dvlop Stability indicing rvrs phas high prformanc liquid chromographic mthod for simultanous stimion of Mupirocin and Mtronidazol in Combind Dosag Form. MATERIALS AND METHODS: Chmicals: Mupirocin and Mtronidazol powdr rfrnc standard (rs) was rcivd as a gift sampl. Th Co - Mupimt granuls containing Mtronidazol (1w/w) and Mupirocin (2w/w) manufacturd and marktd by Fourrts India Labororis pvt ltd was purchasd from local pharmacy. Th HPLC grad mthanol and Actonitril wr purchasd from Mrck spcialtis pvt, Ltd., Mumbai. Instrumntion RP-HPLC instrumntion: Shimadzu LC-20 AT HPLC systm, using SPD-20A UV Dtctor. Th column usd is C18 (25 cm 0.46 cm) Hyprsil BDS. Th tmprur of th column was maintaind room tmprur and th flow r 1.0 n. Th injction volum is 20μl, 230nm was st as a wavlngth and th HPLC run tim was slctd for 7 minuts. phas: Potassium Dihydrogn Phosph buffr and mthanol (adjust to ph 3.5 orthophosphoric acid) in th rion(70:30) v/v was usd for sparion of this drug. Th mobil phas was filttrd through th 0.22μ mmbran filtr aftr sonicion of ach solvnt for 20 min. Slction of dilunts:- Dilunt : Th drug s physical proprtis from th litrur availabl suggstd th th drug is solubl in Mthanol and xprimntal rsults also showd th sam, thus Mthanol was trid for th dilunt and xprimntal rsults producd wr good. Th sam dilunt was applid for th formulions availabl and found sisfactory as th dilunt. RP-HPLC Standard solution: Mupirocin standard stock solution (200 μg/ml): Accurly wightd 20 mg of Mupirocin was transfrrd to a 100 ml volumtric flask. To th volumtric flask ml of mthanol was addd and mixd thoroughly to dissolv. Thn th volum was adjustd to mak up th volum to 100 ml with mthanol. Mtronidazol standard stock solution (100 μg/ml) : Accurly wightd 10 mg of Mtronidazol was transfrrd to a 100 ml volumtric flask. To th volumtric flask ml of mthanol was addd and mixd thoroughly to dissolv. Thn th volum was adjustd to mak up th volum to 100 ml with mthanol. Prparion of standard solution of binary mixturs of Mtronidazol (10 μg/ml) and Mupirocin (20 μg/ml) : Transfr 1 ml of th Mtronidazol stock solution (100 μg/ml) and 1mL from Mupirocin stock solution (200 μg/ml) to 10 ml volumtric flask and volum was mad up to th mark by mobil phas which was usd in particular trials. Prparion of formulion solution: Wigh th Powdr formulion quivalnt to 10 mg of Mtronidazol and 20 mg of Mupirocin and transfr to a 100 ml volumtric flask. Add 60 ml of phas and shak for fw minuts and mad up volum up to th mark with mobil phas. Th solution was filtrd 118
3 through Whman filtr papr no. 42 and first fw drops of filtr wr discardd. Piptt out 1 ml of this solution a dilut to 10 ml with mobil phas. Acid dgradion: On ml of stock solution was transfr in to 10 ml of volumtric flask and to th two ml of 0.1 N HCl solutions was addd and mixd wll. This solution was kpt room tmprur for 4 hrs. Th rsulting solution is thn nutralizd using 0.1N NaOH solution. Th volum is thn mad up using th dilunt to gt 10 μg/ml for Mtronidazol and 20 μg/ml for Mupirocin. Th abov solution thus obtaind is filtrd and thn injctd. Bas dgradion: On ml of stock solution was transfrrd in to 10 ml of volumtric flask and to th two ml of 0.1 N NaOH solutions was addd and mixd wll. This solution was kpt room tmprur for 4 hrs.. Th rsulting solution is thn nutralizd using 0.1N HCL solution. Th volum is thn mad up using th dilunt to gt 10 μg/ml for Mtronidazol and 20 μg/ml for Mupirocin. Th abov solution thus obtaind is filtrd and thn injctd. Oxidiv dgradion: Th oxidiv dgradion was carrid out using 2 ml of 3 H2O2 which is addd to th on ml of stock solution in to 10 ml of volumtric flask. Th rsultant solution was kpt room tmprur for 4 hrs. Th volum is thn mad up using th dilunt to gt 10 μg/ml for Mtronidazol and 20 μg/ml for Mupirocin. Th abov solution thus obtaind is filtrd and thn injctd. Photo Dgradion: Photo Dgradion studis wr prformd undr th UV light. On ml of stock solution was transfrrd in to 10 ml of volumtric flask and kpt undr UV Light for approximly 10hrs. Thn th volum was adjustd with dilunts to gt 10μg/ml for Mtronidazol and 20μg/ml for Mupirocin. RESULT AND DISCUSSION Th mthod was validd for its linarity rang, accuracy, prcision, snsitivity and spcificity. Mthod validion is carrid out as pr ICH guidlins. Linarity Th linarity for Mtronidazol and Mupirocin wr assssd by analysis of combind standard solution in rang of 5-15μg/ml and 10-30μg/ml rspctivly. Corrlion co-fficint for calibrion curv Mtronidazol and Mupirocin was found to b for both. Tabl 1 : Linarity da for Mtronidazol Corrlion () Cofficint > Rgrssion lin quion y = 130.2x Slop Tabl 2 : Linarity da Mupirocin Corrlion () Cofficint > Rgrssion lin quion y = x Slop Thrmal dgradion: Thrmal Dgradion studis wr prformd by taking on ml of stock solution was transfrrd in to 10 ml of volumtric flask. Th volumtric flask was stord in ovn 110 C for 10 hrs. Thn th volum was adjustd with dilunt to gt 10μg/ml for Mtronidazol and 20μg/ml for Mupirocin. Figur 3 Calibrion Curv of Mtronidazol (5-15μg/ml) 119
4 Th da for intraday prcision for Mtronidazol and Mupirocin is shown in tabl 5. Th. for Intraday prcision was found to b for Mtronidazol and for Mupirocin. Intrday prcision: Figur 4 Calibrion Curv of Mupirocin (10-30μg/ml) Rpability: Th da for rpability of pak ara masurmnt for Mtronidazol and Mupirocin, basd on six masurmnts of sam solution Mtronidazol and Mupirocin ar dpictd in tabl 3,& 4. Th RSD for Mtronidazol and Mupirocin was found to b 0.44and 0.36 rspctivly. Sr Tabl 3 Rpability da for Mtronidazol Conc S.D (n=6) ± Tabl 4 Rpability da for Mupirocin Sr Conc S.D (n=6) ± Intraday prcision 0.36 Th da for intraday prcision for Mtronidazol and Mupirocin is shown in tabl 6. Th. for intrday prcision was found to b for Mtronidazol and for Mupirocin. Tabl 5 Intraday prcision da for stimion of Mtronidazol and Mupirocin Mtronidazol ± ± ±13.28 Mupirocin ± ± ±5.78 Tabl 6 Intrday prcision da for stimion of Mtronidazol and Mupirocin Mtronidazol ± ± ±27.62 Mupirocin ± ± ± Lvl () Tabl 7 Rcovry da for Mtronidazol Sampl amount Addd rcovrd Rcovry Man Rcovry ± S.D ± ± ±
5 Lvl () Sampl Tabl 8 Rcovry da for Mupirocin Addd rcovrd Rcovry Man Rcovry ± S.D ± ± ± Tabl 9 Robustnss da for Mtronidazol r (- 0.2 n) r (+ 0.2 n) ph (- 0.2) ph (+0.2) phas (-2) phas( +2) Tabl 10 Robustnss da for Mupirocin r (- 0.2 r (+ 0.2 ph (- 0.2) ph (+ 0.2) phas (-2) phas( +2) n) n) R.S. D Accuracy: Accuracy of th mthod was confirmd by rcovry study from marktd formulion thr lvl of standard addition. Th rsults ar shown in tabl 7 and tabl 8 Prcntag rcovry for Mtronidazol was , whil for Mupirocin, it was found to b in rang of Robustnss: Th ffct of changs was found to b within th accptanc critria as shown in tabl 9 and tabl 10.Th RSD should B lss than 2. LOD and LOQ: Calibrion curv was rpd for fiv tims and th standard dviion (SD) of th intrcpts was calculd.thn LOD and LOQ wr calculd as follows: Limit of Dtction : Limit of Quantizion : Mtronidazol Mupirocin µg/ml µg/ml µg/ml 2.227µg/ml CONCLUSION Th study shows th th dvlopd HPLC Mthod is fast, prcis, spcific, accur and stability indicing. Th stability -indicing mthod rsolvd th drug pak and also th paks of dgradion products formd undr varity of conditions. Th drug was subjctd to strss condition of hydrolysis, oxidion, photolysis and Thrmal dgradion, Considrabl Dgradion was found in alkalin dgradion. Th proposd mthod was succssfully 121
6 applid for th simultanous stimion of both th drugs in commrcial Combind dosag form. Hnc, th mthod is rcommndd for routin quality control analysis of Mtronidazol and Mupirocin in prsnc of dgrad product and othr pharmacutical xcipints prsnt in dosag form. REFERENCES: 1) Drug profil for Mupirocin, spt ) Indian Pharmacopoia, Vol. ΙΙ, Govrnmnt of India, Th controllr of Publicions, Dlhi, (2014), pp , ) Th Unitd Sts Pharmacopoia, 27th Rvision, NF 27, Th Unitd Sts Pharmacopoial Convntion, Inc., Rockvill, MD, (2004), 1489, ) British Pharmacopoia, Vol. II, Stionary Offic, Mdicins and Halthcar Products Rgulory Agncy, London, (2010), & ) Th Unitd Sts Pharmacopoia, 32th Rvision, NF 27, Th Unitd Sts Pharmacopoial Convntion, Inc., Rockvill, MD, (2009), & ) Anuradha Y, Rao PV andthangavalan B, Mthod Dvlopmnt And Validion For Simultanous Estimion Of Mupirocin AndMtronidazol In Combind Dosag Form By RP-HPLC World j. pharm. and pharm. rs., 2015, 4(7), ) Pradhan PK, Pl J, Pl M andmavani D, RP- HPLC And UV Spctroscopy Mthod Dvlopmnt And Validion For Simupirocinltanous Estimion Of Mtronidazol And Mupirocinpirocin InThir Combind Dosag Form Invnti, ) British Pharmacopoia, Vol. I & II, Stionary Offic, Mdicins and Halthcar Products Rgulory Agncy, London, (2009), , ) Drug profil for Mtronidazol, spt ronidazol. 6) Indian Pharmacopoia, Vol. ΙΙ, Govrnmnt of India, Th controllr of Publicions, Dlhi, (2010), &
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