Asin Journl of Phrmceuticl Eduction nd Reserch Vol -7, Issue-2, April-June 2018 ISS: 2278 7496 RESEARCH ARTICLE Impct Fctor: 5.019 DEVELOPMET AD VALIDATIO OF RP-HPLC METHOD FOR SIMULTAEOUS ESTIMATIO OF AMOXICILLI TRIHYDRATE, METROIDAZOLE AD FAMOTIDIE idhi Jin Singhi*, Sumn Rmteke School of Phrmceuticl Sciences, Rjiv Gndhi Proudyogiki Vishwvidyly, Bhopl *Corresponding Author s E mil: nidhinidhijn25@gmil.com Received 15 Mr. 2018; Revised 18 Mr.2018; Accepted 28 Mr. 2018, Avilble online 15 Apr. 2018 ABSTRACT The objective of the current study ws to develop nd vlidte simple, ccurte, precise nd rpid reversed-phse HPLC method for simultneous estimtion of Amoxicillin trihydrte (AMOX), Metronidzole (METRO) nd Fmotidine (FAMO). The chromtogrphic seprtion of AMOX, METRO nd FAMO were chieved on RP-HPLC hving Lun C-18-ODS bonded column of length 250mm using UV detection t 267 nm. The optimized mobile phse consisted of mixture of 0.03M disodium hydrogen phosphte buffer cetonitrile (93:7, v/v) djusted to ph 6.5 t flow rte of 1.5 ml/min. The retention times were 2.560, 3.657 nd 6.983 min for AMOX, FAMO nd METRO respectively. The proposed method provided liner responses within the concentrtion rnges of 0-50µg/ml for Amoxicillin trihydrte & Metronidzole nd 0-30µg/ml for Fmotidine. The LOD vlues were 0.0252, 0.0098 nd 0.0288 µg/ml nd LOQ vlues were 0.0765, 0.0298 nd 0.0875µg/ml for Amoxicillin trihydrte & Metronidzole nd Fmotidine, respectively. High recovery nd low % coefficient of vrition (COV) reveled the relibility of the method for quntittive study of the three drugs in combined dosge form. Keywords: Amoxicillin trihydrte, Metronidzole, Fmotidine, Simultneous estimtion, Reversedphse HPLC method. ITRODUCTIO Helicobcter pylori (H. pylori) is spirl-shped, Grm-negtive bcterium tht chroniclly infects the gstric mucos of >50% of the humn popultion, cusing chronic inflmmtion of the stomch nd development of gstroduodenl diseses, such s gstritis, peptic ulcer nd gstric cncer 1. The most widely recommended tretment in interntionl guidelines for the erdiction of H. pylori is combintion of two ntibiotics with n cid-suppressing gent for t lest 14 dys 2. Amoxicillin trihydrte (AMOX), 6(R)-6-{ -D- (4- hydroxyl phenyl) glycyl mino} penicillnic cid trihydrte is n orlly bsorbed, semi-synthetic brod-spectrum ntimicrobil drug 3-4. Metronidzole (METRO), 1-(β-hydroxy-ethyl)-2-methyl-5-nitroimidzole is used s ntiprotozol nd ntibcteril gent 5-6. Fmotidine (FAMO), (minosulfonyl)-3 [[[2-[(di-minomethylene) mino] 4-trizolyl] methyl] thio] propnimidmide is potent, competitive nd reversible inhibitor of histmine ction t the H2 receptor 7-8. The combintion of Amoxicillin trihydrte, Metronidzole nd Fmotidine, is now
widely used in stndrd erdiction tretment of gstric nd duodenl ulcers, which re ssocited with H. pylori infection. These triple therpies re provided to be effective in clinicl ppliction 9-11. Combintion of three drugs hs shown more effective ginst the peptic ulcer cused by H.pylori. Till dte there is no HPLC method for the simultneous estimtion of these three drugs combintion. Extensive literture survey suggested tht formultion contining these three drugs in combintion hs not been reported so fr nd hence the method of nlysis is lso not vilble 12-15. The present study revels simple, specific, precise nd rpid RP-HPLC method for simultneous estimtion of Amoxicillin trihydrte, Metronidzole nd Fmotidine in combined dosge form. The developed method is further vlidted per ICH guidelines Q2A nd Q2B 16-18. HO H 2 C C H O H O H H S CH 3 CH 3 C OOH. 3H 2 O Amoxicillin trihydrte CH 2 CH 2 OH O 2 CH 3 Metronidzole H 2 H 2 C CH 2 SCH 2 - CH 2 - C - H 2 S SO 2 H 2 Fmotidine Figure 1. Chemicl structures of Amoxicillin trihydrte (AMOX), Metronidzole (METRO) nd Fmotidine (FAMO).
MATERIAL AD METHODS Regents nd chemicls The gift smples of drugs i.e. Amoxicillin trihydrte, Metronidzole nd Fmotidine were provided by Lpiz Phrm, Sgr, Khndelwl lbortory, Mumbi nd Lupin Reserch Prk, Pune, respectively. Disodium hydrogen phosphte, sodium hydroxide, cetonitrile nd ll other chemicls were purchsed from Himedi lbs, USA. All the chemicls of HPLC nd nlyticl grdes were used without ny further purifiction. All the excipients used for the development of plcebo formultions were obtined from commercil sources nd were used s such. Double distilled wter ws used during entire HPLC procedure. Equipment/instrumenttion Shimdzu LC 10 AT VP series HPLC ws used hving photodiode rry detector. Lun C-18-ODS bonded column of length 250 mm nd n inter dimeter 4.6 mm ws selected for the nlysis. The prticle size of the sttionry phse ws 5 µm. The mobile phse ws degssed nd filtered (0.45 µm, Milipore) mixture of 0.03M disodium hydrogen phosphte buffer cetonitrile (93:7, v/v) djusted to ph 6.5 t flow rte of 1.5 ml/min. Preprtion of Stndrd Stock Solutions nd Smple Preprtions The stndrd stock solutions of Amoxicillin trihydrte, Metronidzole nd Fmotidine were prepred by dissolving 50mg of ech drug in 100ml of mobile phse seprtely. From the bove solution 10mL of solution ws tken nd diluted to 50ml with the sme to get solution contining 100 µg/ml of ech drug. From the stock solutions, eight working stndrd solutions for three drugs hving concentrtion 5, 10, 15, 20, 25, 30, 35, 40, 45, 50µg/ml were prepred in mobile phse nd their re ws noted by injecting 20µL into the system. After tht clibrtion curves were plotted between concentrtion ginst their respective re for AMOX, Metronidzole nd Fmotidine seprtely. From the clibrtion curve it ws found tht AMOX nd Metronidzole hve linerity rnge between 0-50µg/ml wheres Fmotidine hs rnge between 0-30µg/ml. Preprtion of Mixed Stndrd Solutions Five mixed stndrds solutions with concentrtion of Amoxicillin trihydrte, Metronidzole nd Fmotidine in g/ml of 5:25:30, 10:20:25, 15:15:20, 20:10:15, 25:5:10 were prepred in mobile phse by diluting pproprite volumes of the stndrd stock solutions. The solutions were loded in the injector fitted with 20 µl fixed volume loop nd re ws recorded.
Method Vlidtion Linerity For checking linerity stndrd stock solutions of Amoxicillin trihydrte, Metronidzole nd Fmotidine were prepred by dissolving 50mg of ech drug in 100ml of mobile phse seprtely. From the bove solution 10ml of solution ws tken nd diluted to 50ml with the sme to get solution contining 100 µg/ml of ech drug. From the stock solutions, ten working stndrd solutions for three drugs hving concentrtion 5, 10, 15, 20, 25, 30, 35, 40, 45, 50µg/ml were prepred in mobile phse nd their re ws noted by injecting 20µL into the system. After tht clibrtion curves were plotted between concentrtion ginst their respective re for Amoxicillin trihydrte, Metronidzole nd Fmotidine, seprtely. Accurcy nd precision Accurcy is performed to check similrity of results obtined by nlyticl vlue to the true vlue. The precision is defined s degree of this similrity. To check the ccurcy of the proposed methods, recovery studies were crried out t 80, 100, nd 120% of the stndrd concentrtion s per ICH guidelines 19, 20. The recovery study ws performed three times t ech level. Intermedite Precision- (Inter-dy nd Intr-dy precision) Intermedite Precision of the method ws inter-dy nd intr-dy nlysis i.e. the nlysis of formultion ws repeted six times in the sme dy nd on three successive dys 21. Limit of Detection (LOD) nd Limit of Quntittion (LOQ) The LOD nd LOQ of Amoxicillin trihydrte, Metronidzole nd Fmotidine determined by clibrtion stndrd method. LOD nd LOQ were clculted using the following equtions; LOD = 3.3 (σ/s) nd LOQ = 10 (σ /S), where σ is stndrd devition (SD) of the y-intercept of clibrtion curve nd S is slope of regression eqution 22. For LOD nd LOQ, 1µg/ml of solution of three drugs ws prepred from stndrd stock solution contining 100µg/ml by diluting pproprite volume with mobile phse. Five stndrd solutions for Amoxicillin trihydrte hving concentrtion 0.4, 0.8, 1.0, 1.2, 1.4 µg/ml nd for Metronidzole nd Fmotidine hving concentrtion 0.8, 1.0, 1.2, 1.4, 1.6 µg/ml were prepred in mobile phse from 1 µg/ml of solution re ws noted.
Results nd discussion Linerity From the clibrtion curve (Figure no. 1, 2 nd 3) it ws found tht Amoxicillin trihydrte nd Metronidzole hve linerity rnge between 0-50µg/ml wheres Fmotidine hs rnge between 0-30µg/ml. For ech drug, pproprite dilutions of stndrd stock solutions were ssyed s per the developed methods. The liner regression eqution for three drugs ws; For AMOX Y= 12628x 1465.4 (r 2 =0.9981) For METRO Y= 18837x + 4117.8 (r 2 =0.9987) For FAMO Y = 22925x 7449.8 (r 2 = 0.9975) 700000 P e k r e 600000 500000 400000 300000 200000 100000 0 y = 12648x - 2149.3 R 2 = 0.9975 0 10 20 30 40 50 60 Concentrtion (µg/ml) Figure 1: Clibrtion Curve of Amoxicillin Trihydrte
P e k r e 1000000 900000 800000 700000 600000 500000 400000 300000 200000 100000 0 y = 18782x + 6039.5 R 2 = 0.9982 0 10 20 30 40 50 60 Concentrtion (µg/ml) Figure 2: Clibrtion Curve of Metronidzole P e k r e 800000 700000 600000 500000 400000 300000 200000 100000 0 y = 23223x - 13906 R 2 = 0.9965 0 5 10 15 20 25 30 35 Concentrtion ( g/ml) Figure 3: Clibrtion Curve of Fmotidine
Specificity The Figure no. 4 shows two-dimensionl chromtogrm for three drugs indicting no interference between ll three drugs t 267 nm. Good seprtion is seen s the retention times were 2.560, 3.657 nd 6.983 min for Amoxicillin trihydrte, Metronidzole nd Fmotidine, respectively. Although there is less difference between Amoxicillin trihydrte nd Fmotidine but still peks re cler distinguished which ws further supported by vlidtion dt. The totl smples were run for 10 min to llow lte eluting pek. The 10 min run is sufficient for ny smple nlysis to llow nlysis of lrge no. of smple in less time 23. Figure 4: Chromtogrm of AMOX, METRO nd FAMO in Smple Solution nd its Retention Time t 267nm. Accurcy nd precision The dt obtined (Tble no. 1) shows tht % recovery of ll drugs lies between 99.50-100.50 %, which proves tht developed method is ccurte nd lies well within recommended tolernce of 80-115 %. It is considered tht method is vlidted when its ccurcy is within ± 15% nd precise when COV is below 15% 24.
Tble 1 Anlysis of the results of recovery experiments Amount tken (µg/ml) Amount dded t (µg/ml) % Recovery AMOX METRO FAMO % AMOX METRO FAMO AMOX METRO FAMO 1 20 10 4 16 8 3.2 99.50 ± 0.17 99.80 ± 0.10 100.00 ±0.22 2 20 10 4 80% 16 8 3.2 100.10 ± 0.11 100.40 ± 0.24 100.20 ± 0.16 3 20 10 4 16 8 3.2 100.00 ± 0.25 99.60 ± 0.13 100.50 ± 0.10 1 20 10 4 20 10 4 99.50 ± 0.18 99.70 ± 0.19 100.00 ± 0.28 2 20 10 4 100% 20 10 4 100.20 ± 0.12 99.90 ± 0.25 100.50 ± 0.31 3 20 10 4 20 10 4 100.30 ± 0.28 99.70 ± 0.16 99.50 ± 0.19 1 20 10 4 24 12 4.8 99.00 ± 0.22 100.30 ± 0.20 99.75 ± 0.26 2 20 10 4 120% 24 12 4.8 100.00 ± 0.16 100.00 ± 0.07 100.30 ± 0.12 3 20 10 4 24 12 4.8 99.50 ± 0.35 99.50 ± 0.28 99.80 ± 0.10 Intermedite Precision- (Inter-dy nd Intr-dy precision) Precision ws determined by repetbility (intr-dy precision) nd intermedite precision (inter-dy precision) nd ws expressed s the reltive stndrd devition (RSD) of series of mesurements. The repetbility ws evluted by ssying six smples t the sme concentrtion (12.0 µg/ml) during the sme dy. The intermedite precision ws evluted by repeting the studies on three different dys nd compring the obtined results. The dt of intr-dy nd inter-dy precision nd ccurcy for the method re listed in Tble 2. Intermedite precision study expresses the intr-dy nd inter-dy precision ws determined by ssy of the smple solution on the sme dy t different time intervls nd on different dys, respectively. The dt obtined (tble no. 2) shows tht for ll the methods, % coefficient of vrition (COV) ws not more thn 2.0% which indictes well intermedite precision.
Tble 2 Results of intr-dy nd inter-dy precision Intrdy precision Inter dy precision % Retention % Retention Time AMOX METRO FAMO Dy AMOX METRO FAMO After 1hr 99.50±0.17 100.50±0.17 100.25±0.17 First dy 99.10±0.25 100.10±0.10 100.20±0.22 After 2hr 99.63±0.17 100.00±0.17 100.40±0.17 Second dy 99.20±0.22 99.90±0.12 99.95±0.26 After 3hr 99.10±0.17 100.25±0.17 100.50±0.17 Third dy 99.35±0.12 99.70±0.13 100.20±0.22 After 4hr 99.20±0.17 100.10±0.17 100.35±0.17 After 5hr 99.50±0.17 100.20±0.17 100.40± After 6hr 99.30±0.17 100.30±0.17 100.50±0.17 Men 99.37 100.22 100.40 Men 99.21 99.90 100.08 SD 0.2040 0.1724 0.0948 SD 0.2000 0.1258 0.2158 %COV 0.2052 0.1720 0.0944 % COV 0.2015 0.1259 0.2156 AMOX: Amoxicillin trihydrte, METRO: Metronidzole, FAMO: Fmotidine, S.D.: Stndrd devition, COV: Coefficient of vrition, Vlues represent men ± SD (n = 3) Limit of Detection (LOD) nd Limit of Quntittion (LOQ) The Dt for LOD nd LOQ for different drugs re shown in tble no 3, 4, nd 5. The LOD vlues were 0.0252, 0.0098 nd 0.0288 µg/ml nd LOQ vlues were 0.0765, 0.0298 nd 0.0875µg/ml for Amoxicillin trihydrte, Metronidzole nd Fmotidine, respectively. High recovery nd low % COV reveled the relibility of the method for quntittive study of three drugs in combined dosge form. LOD vlues of clibrtion curves indictes the lowest concentrtion of nlyte(s) in smple tht cn be detected under stted experimentl conditions nd LOQ vlues of clibrtion curves indictes the lowest concentrtion of nlyte(s) in smple tht cn be determined with cceptble precision nd ccurcy under the stted experimentl conditions 25.
Tble 3 HPLC nlysis of AUC of AMOX Std. conc. (µg/ml) Replicte 1 2 3 4 5 Men 0.6 5378±117 5278±122 5478±119 5270±105 5288±109 5278 0.8 10553±123 10753±102 10653±127 10650±111 10656±0124 10653 1.0 13416±115 13216±132 13316±106 13300±118 13332±135 13316 1.2 15779±103 15979±109 15850±114 15908±126 15879±124 15879 1.4 18625±119 18425±108 18500±122 18550±131 18525±128 18525 Anlyticl prmeters LOD 0.0252µg/ml LOQ 0.0765µg/ml SD 120.968 Slope 15799.8 Vlues represent men ± SD (n = 3) Tble 4 HPLC nlysis of AUC of METRO Stndrd concentrtion (µg/ml) Replicte 1 2 3 4 5 Men 0.8 12433±337 12633±265 12533±279 12733±310 12333±283 12533 1.0 15660±268 15760±301 15560±254 15460±279 15860±281 15660 1.2 20692±290 20892±264 20792±307 20592±291 20992±279 20792 1.4 24357±304 24157±294 24257±306 24457±293 24057±300 24257 1.6 27822±313 27622±299 27922±280 27522±291 27722±297 27722 Anlyticl prmeters LOD 0.0098µg/ml LOQ 0.0298µg/ml SD 296.985 Slope 19488 Vlues represent men ± SD (n = 3)
Tble 5 HPLC nlysis of AUC of FAMO Stndrd concentrtion (µg/ml) Replicte 1 2 3 4 5 Men 0.8 14392 ± 168 14192 ± 180 14292 ± 153 14200 ± 193 14492 ± 172 14292 1.0 17812 ± 175 17212 ± 192 17512 ± 186 17712 ± 155 17312 ± 182 17512 1.2 21000 ± 181 21028 ± 179 21514 ± 192 20514 ± 188 21014 ± 173 21014 1.4 25604 ± 184 25404 ± 183 25504 ± 172 25304 ± 191 25704 ± 177 25504 1.6 30247 ± 191 30047 ± 170 30147 ± 182 30157 ± 179 30137 ± 183 30147 Anlyticl prmeters LOD 0.0288µg/ml LOQ 0.0875µg/ml SD 173.534 Slope 19829.4 Vlues represent men ± SD (n = 3) Conclusion The proposed RP-HPLC method is rpid, sensitive, nd reproducible, llows ccurte, precise nd relible mesurement of Amoxicillin trihydrte, Metronidzole nd Fmotidine simultneously in combined dosge form. The RSD for ll prmeters ws found to be less thn 2%, which indictes the vlidity of method. Thus, the developed method cn be used for routine quntittive simultneous estimtion of AMOX, Metronidzole nd Fmotidine in combined dosge form. Acknowledgement: We grtefully cknowledge the Lpiz Phrm, Sgr, Khndelwl lbortory, Mumbi nd Lupin Reserch Prk, Pune, for supplying gift smples of Amoxicillin trihydrte, Metronidzole nd Fmotidine, respectively to crry out the study. References 1. Petersen AM nd Krogfett KA. Helicobcter pylori: n invding microorgnism? A review; FEMS Immunology & Medicl Microbiology. 2003; 36: 117-126. 2. Shiotni A nd Grhm DY. Pthogenesis nd therpy of gstric nd duodenl ulcer disese; Medicl Clinics of orth Americ. 2002; 86:1447-1466. 3. Toms A. The mechnism of the irreversible ntimicrobil effects of penicillins: how the bet-lctm ntibiotics kill nd lyse bcteri; Ann Rev. of Microbiol, 1979; 33: 113.
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