HPLC method development and validation for simultaneous estimation of Olmesartan Medoxomil, Hydrochlorothiazide and Amlodipine Besylate tablets
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1 Avilble online t Scholrs Reserch Librry Der Phrmci Lettre, 2015, 7 (5): ( ISSN USA CODEN: DPLEB4 HPLC method development nd vlidtion for simultneous estimtion of Olmesrtn Medoxomil, Hydrochlorothizide nd Amlodipine Besylte tblets Ayyknnu Arumugm Npoleon 1 *, Gngdhr Angjl 2 nd Rj. A. 1 1 Phrmceuticl Chemistry Division, School of Advnced Sciences, VIT University, Vellore, Tmil Ndu, Indi 2 Orgnic Chemistry Division, School of Advnced Sciences, VIT University, Vellore, Tmil Ndu, Indi ABSTRACT In the present work for the determintion of Olmesrtn Medoxomil, Hydrochlorothizide nd Amlodipine simple, rpid nd precise high performnce liquid chromtogrphy procedure hs been developed nd vlidted. The seprtion nd quntifiction were chieved on Cyno C18 column using mobile phse of filtered nd degssed mixture of buffer nd cetonitrile in the rtio of 830:170 with of nlytes t 237 nm. Buffer ws prepred by dissolving 3.65 g of mmonium cette in 1000 ml of wter nd the ph ws djusted to 7.0 with sodium hydroxide t flow rte of 1.5 ml/min. The method showed good linerity with different concentrtions ner to , nd for Olmesrtn Medoxomil, Hydrochlorothizide nd Amlodipine respectively. The recovery (Averge men & RSD) were chieved s , 1.01 for Olmesrtn Medoxomil, , 0.26 for Hydrochlorothizide nd , 0.26 for Amlodipine respectively. The precision (Averge men & RSD) were chieved s 99.9, nd for Olmesrtn Medoxomil, Hydrochlorothizide nd Amlodipine respectively. Keywords: HPLC, Amlodipine Besylte, Olmesrtn Medoxomil, Hydrochlorothizide. INTRODUCTION The totl number of drugs relesed in to the mrket is incresing drmticlly over the pst decdes cross the globe. Most of the drugs re either the prtil structurl modifiction of the existing drugs or new entities [1]. In some cses there will be time lg from the dte of drug introduction in to the mrket to the dte of its inclusion in phrmcopoeis. This usully hppens s result of uncertinity in continuous wider usge of these drugs, toxicity reports, development of resistnce with lredy existing methods, or it my not provide enough dequte sensitivity. In these conditions, sufficient stndrd nlyticl procedures my not be vilble in the phrmcopoeis due to ptent regultions nd moreover there my not be suitble method for prticulr nlyte in the specific smple mtrix [2-3]. Therefore it becomes necessry to develop new nlyticl methods for such drugs. Methods for drug nlysis cn be developed provided one should possess sound knowledge bout the nture of the smple with respect to its polrity, moleculr weight, solubility prmeter nd ionic chrcter. Now dy the development of method for drug nlysis is usully bsed on prior rt or existing literture using the sme quite similr instrumenttion. It is very rre tht n HPLC method is developed tht does not in some wy relte to existing literture-bsed methods. The development of ny new or improved method usully tilors existing pproches nd instrumenttion to the current nlyte s well s to the finl requirements of the method. 182
2 HPLC is considered to be most suitble nlyticl method for phrmceuticls nd remins s useful tool in future despite further dvnces in nlyticl chemistry becuse of severl overhelming dvntges for the solution of mny problems. These dvntges include speed, simplicity, specificity nd sensitivity. Literture studies revel tht there re nlyticl methods vilble for determintion of Amlodipine, Olmesrtn nd Hydrochlorothizide in combintion with other drugs [4-18] nd only few nlyticl methods vilble for simultneous estimtion of Amlodipine, Olmesrtn nd Hydrochlorothizide in combined form. Doshi et l., hve reported vlidted reverse phse HPLC method for simultneous estimtion of ll the three drugs by using Buffer:Acetonitrile (contining 10% Tetrhydrofurn) s mobile phse [19]. Nlluri et l., hve reported development nd vlidtion of reverse phse HPLC-PDA method for simultneous estimtion of Amlodipine, Olmesrtn nd Hydrochlorothizide in combined form by using 0.1% v/v formiccid:cetonitrile s mobile phse [20]. Although the methods shows optimum retention time it fils to provide detiled explntion on stbility control nd lcks vlidtion in terms of stress conditions like cid, bse, het, oxidtion, light nd UV. Also the pek shpes nd resolution of the bove two methods were lso not pproprite. Kumr et l., hve reported ultr performnce liquid chromtogrphy method development for simultneous estimtion of ll the three drugs by using sodium perchlorte:cetonitrile s mobile phse [21]. This method does not provide complete vlidtion of ll the three drugs in terms of its system suitbility nd robustness. Moreover the method developed ws not completely reproducible nd the pek resolution ws not good. Hence in the present work it ws plnned to develop stbility indicting HPLC method, which would be precise, ccurte, simple, reproducible nd relible for mrketed drug combintion of Olmesrtn Medoxomil, Hydrochlorothizide nd Amlodipine Besylte tblets. The objective thus, ws to subject this new tblet formultion to HPLC nlysis by different tril methods nd finlly rrive t suitble method for the determintion of the drugs therein. The present work ws minly confined to the estimtion of content of ctive ingredient with good resolution nd optimum retention time simultneously covering the vlidtion ssys completely s per ICH guidelines [22-23]. Olmesrtn Medoxomil belongs to the clss of medicines clled ngiotensin II receptor ntgonists to tret high blood pressure nd vilble in 5, 20 nd 40 mg tblet. Olmesrtn is prodrug hving one tetrzole nd one imdzole group. It usully blocks the binding of ngiotensin-ii to the AT 1 receptors in vsculr muscle thereby reduces vsoconstriction nd secretion of ldosterone which utomticlly lowers blood pressure by producing vsodiltion nd decresing peripherl resistnt [24]. Amlodipine Besylte is long-cting clcium chnnel blocker generlly used s n nti-hypertensive nd in the tretment of ngin. It inhibits clcium ion influx cross cell membrne selectively with more effect on smoother muscles thn on crdic muscles thereby reducing peripherl vsculr resistnt nd blood pressure. Hydrochlorothizide belongs to the thizide clss of diuretics, minly cting on the kidneys to reduce sodium rebsorption in the distl convoluted tubule. It helps in the rebsorption of clcium in distl convoluted tubule simultneously lowering peripherl vsculr resistnce [25]. MATERIALS AND METHODS HPLC instrument model nme: SPD-M10Avp (Shimdzu) PDA Detector. Softwre: Clss VP. Chemicls: Acetonitrile HPLC Grde (Quligens pvt.ltd), Methnol (Quligens pvt.ltd), Ammonium cette AR (E- Merk Ltd,.Indi), Sodium hydroxide AR (E-Merk Ltd,.Indi), Milli-Q-wter (Rnkem) Reference stndrd purity: Olmesrtn Medoxomil (99.92 %), Hydrochlorothizide (100 %), Amlodipine Besylte (99.22 %). Ech film coted tblet contins Olmesrtn Medoxomil (20 mg), Hydrochlorothizide (12.5 mg) BP, nd Amlodipine Besylte 5 mg BP equivlent to Amlodipine (Micro Lbs Ltd). Anlyticl method development Tril Method: 1 As per in-house method (product relese specifiction) combintion of Olmesrtn Medoxomil, Amlodipine Besylte nd Hydrochlorothizide. Chromtogrphic conditions Mobile phse composition: Buffer: Acetonitrile: Methnol (70:10:30) [Buffer M Ammonium cette in 1000 ml of wter djust the ph to 7.0 with NOH. Column : Cyno C18, mm, 5 µm (thermo hypersil column) Detector : 225 nm 183
3 Diluent : Acetonitrile : Wter (80:20) Flow rte : 1.5 ml/min Injection volume : 10 µl Concentrtion of stndrd: Olmesrtn Medoxomil (0.1 mg/ml), Hydrochlorothizide ( mg/ml) nd Amlodipine (0.025 mg/ml) Inference: Amlodipine peks completely merged with Olmesrtn peks nd pek resolution ws not good. So ttempt ws tken to increse the difference in retention time by incresing the proportion of cetonitrile which resulted in pek brodening. Fig.1A, Tble:1 Tble 1 S.No Pek Nme Retention Time (min) Are Reltive Are (mau X min) (%) Resolution (EP) Asymmetry (EP) Pltes (EP) 1. Hydrochlorothizide Olmesrtn Amlodipine Totl Tril Method: 2 Different mobile phse composition Chromtogrphic conditions Buffer: Acetonitrile: Methnol (60:20:20) [Buffer M Ammonium cette in 1000 ml of wter] the ph ws djusted to 7.0 with NOH Column : Cyno C18, mm, 5 µm (thermo hypersil column) Detector : 225 nm Diluent : Acetonitrile: Wter (80:20) Flow rte : 1.5 ml/min Injection volume : 10 µl Concentrtion of stndrd: Olmesrtn Medoxomil (0.1 mg/ml), Hydrochlorothizide ( mg/ml) nd Amlodipine (0.025 mg/ml). Inference: Pek resolution ws not good but decresing the volume of Buffer nd methnol some extend resulted in brodening of peks. Fig.1B, Tble: 2. Tble 2 S.No Pek Nme Retention Time (min) Are Reltive Are (mau X min) (%) Resolution (EP) Asymmetry (EP) Pltes (EP) 1. Hydrochlorothizide n Olmesrtn n Amlodipine n Totl Tril Method: 3 All the chromtogrphic conditions remin sme except the buffer:acetonitrile:methnol (50:40:10) Inference: Pek brodening is observed. Fig.1C, Tble: 3. Tble 3 S.No Pek Nme Retention Time (min) Are Reltive Are (mau X min) (%) Resolution (EP) Asymmetry (EP) Pltes (EP) 1. Hydrochlorothizide Olmesrtn Amlodipine Totl Tril Method: 4 Chromtogrphic conditions Mobile phse composition: 184
4 Buffer : Acetonitrile (87:13) [Buffer M Ammonium cette in 1000 ml of wter] the ph ws djusted to 7.0 with NOH Column : Cyno, mm, 5 µm (thermo hypersil column) Detector : 237 nm Diluent : Acetonitrile : Wter (80:20) Flow rte : 1.5 ml/min Injection volume : 10 µl Concentrtion of stndrd: Amlodipine (0.025 mg/ml) Olmesrtn Medoxomil (0.1 mg/ml), Hydrochlorothizide ( mg/ml) nd Inference: The mobile phse composition gve good resolution nd optimum retention time with pproprite tiling fctor (< 2). The detected wve length ws selected s nm where the peks height of the drug ws cceptble. Fig.1D, Tble: 4. Tble 4 S.No Pek Nme Retention Time (min) Are Reltive Are (mau X min) (%) Resolution (EP) Asymmetry (EP) Pltes (EP) 1. Hydrochlorothizide Olmesrtn Amlodipine Totl RESULTS AND DISCUSSION Method of Anlysis vlidted Chromtogrphic system: HPLC : Shimdzu HPLC with PDA or UV detector nd Clss VP softwre Anlyticl Column : Cyno C18, mm, 5 µm (thermo hypersil column) Detector wve length : 237 nm Diluent : Acetonitrile : Wter (80:20) Flow rte : 1.5 ml/min Injection volume : 10 µl Column temperture : Ambient Buffer: g of Ammonium Acette ws dissolved in 1000 ml of wter nd the ph ws djusted to 7.0 with NOH solution nd filtered. Mobile Phse: Buffer nd Acetonitrile were tken in the rtio of 830:170, mixed well nd degssed. Diluent: Acetonitrile nd Wter ws tken in the rtio of 80: N HCl Preprtion: 8.5 ml of Conc. HCl ws dded to bout 500 ml of wter mixed well nd mde up to 1000 ml with wter. Stndrd Preprtion: A weighed quntity of bout 100 mg of Olmesrtn Medoxomil working stndrd, 62.5 mg of Hydrochlorothizide nd 25 mg of Amlodipine Besylte ws trnsferred ccurtely to 100 ml volumetric flsk to this 15 ml of 0.1 N HCl ws dded nd kept in sonictor to dissolve the contents nd mde up to the volume with diluents. 5 ml of the bove solution ws diluted to 50 ml with diluent nd the resultnt solution ws filtered through 0.45 µ membrne filter. 185
5 Smple Preprtion: About 20 tblets ws crushed into fine powder, nd equivlent tblet powder 100 mg of Olmesrtn Medoxomil ws weighed nd tken into 100 ml volumetric flsk to this 15 ml of 0.1 N HCl ws dded nd kept in sonictor to dissolve the contents nd mde up to the volume with diluents. 5 ml of the bove solution ws diluted to 50 ml with diluent nd the resultnt solution ws filtered through 0.45 µ membrne filter. System suitbility The stndrd preprtion ws chromtogrphed nd pek responses were recorded s directed under procedure. The theoreticl pltes for Olmesrtn pek, Hydrochlorothizide pek, Amlodipine pek should not be less thn 2000, tiling fctor should not be more thn 2.0 nd percent reltive stndrd devition for five replicte injections of stndrd preprtion should not be more thn 2.0 %. Procedure 10 µl of stndrd preprtion nd smple preprtion ws injected sepertely into the chromtogrph nd pek responses for mjor peks were mesured. System Precision System precision were performed dily during entire vlidtion of the method by using following two methods. Repetbility The repetbility of the nlyticl method ws estblished by estimting the ssy for six different smple preprtions of sme btch. Assys for ll the six smple preprtions were clculted nd their %RSD of 6 ssys were tbulted. Theoreticl pltes obtined for Olmesrtn, Hydrochlorothizide nd Amlodipine peks re 2135, 2466 nd 2308 respectively tbulted in Tble: 5 Intermedite Precision (Ruggedness) The ruggedness of the nlyticl method ws estblished by estimting the ssy for six different smple preprtions of sme btch by different nlyst using different HPLC system with different similr column on different dy. The test method meets the criterion for system precision nd the results were tbulted in Tble :5b Selectivity The selectivity of the method demonstrted by injecting the blnk solution (mobile phse), Plcebo preprtion (Excipients), individul Olmesrtn Medoxomil working stndrd (WS) preprtion, Hydochlorothizide WS preprtion nd Amlodipine Besylte WS preprtion, smple preprtion nd mixture of Olmesrtn Medoxomil, Hydrochlorothizide nd Amlodipine Besylte WS solution. Tble : 6-6d Tble 5 System Precision- Repetblity Injection No Are Olmesrtn Hydrochlorothizide Amlodipine Besylte Men SD % RSD b Stndrd Devition; b Reltive Stndrd Devition 186
6 Tble 5b Intermedite Precision Injection No Are Olmesrtn Hydrochlorothizide Amlodipine Besylte Men SD % RSD b Stndrd Devition; b Reltive Stndrd Devition Tble 6 Drug product specificity- Olmesrtn Olmesrtn Smple Pek purity Unstressed Acid Bse ND - Oxidtion Het Light UV No Dt Tble 6b Drug product specificity- Hydrochlorothizide Hydrochlorothizide Smple Pek purity Unstressed Acid Bse Oxidtion Het Light UV Tble 6c Drug product specificity- Amlodipine Amlodipine Smple Pek purity Unstressed Acid ND - Bse Oxidtion Het Light UV No Dt Tble 6d Specificity of smple nd stndrd Olmesrtn Hydrochlorothizide Amlodipine RT in Indin Injection RT in Std RT in Smple Plcebo No Interference Bnk No Interference Forced degrdtion studies Forced degrdtion studies were performed to estblish stbility indicting nture of the ssy method. Tblet smple, Olmesrtn Medoxmil, Hydrochlorothizide, Amlodipine Besylte nd Plcebo (Excipients) to following stress conditions to undergo degrdtion. Tble:
7 Tble 7 Forced degrdtion studies: Vrious stress conditions Smple stress condition Description of stress condition Stress time Acid degrdtion 5N HCl 2 h Bse degrdtion 5N NOH 2 h Oxidtion degrdtion 20% v/v Hydrogen Peroxide 1 h Photo degrdtion Direct exposure to sunlight 12 h Degrdtion by UV Exposed to UV light 120 h Therml degrdtion 105 C 24 h Interference studies The smple solvent nd plcebo solution were injected to demonstrte non-interference. Olmesrtn Medoxomil, Hydrochlorothizide nd Amlodipine Besylte peks were identified by their retention times. Pek purity index for the nlytes of interest were generted. Olmesrtn, Hydrochlorothizide nd Amlodipine peks re free of interference in ll the stress studies where s the nlyticl method is sensitive to the degrdtion (for Olmesrtn, Hydrochlorothizide nd Amlodipine) nd sensitive to bse degrdtion (for Olmesrtn lone). Henceforth the method is selective to Olmesrtn, Hydrochlorothizide nd Amlodipine nd their degrdtion production. Degrdtion product peks did not interfere with the intct min pek, so this method is stbility indicting method. Fig. 3A-3L Linerity Linerity of the nlyticl method ws demonstrted by tking series of solution contining Olmesrtn Medoxomil, Hydrochlorothizide nd Amlodipine Besylte t concentrtion in the rnge % were nlyzed to determine the linerity of the method. Ech injection ws injected in duplicte nd the results were presented in Tble: 8, 8b nd 8c. Fig.4-6. A plot ws drwn between the concentrtions versus pek response of Olmesrtn, Hydrochlorothizide, Amlodipine. The test method ws demonstrted to be liner for ll the three drugs in the concentrtion rnges listed bove. Tble 8 Linerity- Olmesrtn Olmesrtn % Concentrtion Actul Conc ppm Avg Are 70% % % % % % % Tble 8b Linerity- Hydrochlorothizide Hydrochlorothizide % Concentrtion Actul Conc ppm Avg Are 70 % % % % % % % Corr Coeff R Correltion Coefficient 188
8 Tble 8c Linerity- Amlodipine Amlodipine % Concentrtion Actul Conc ppm Avg Are 70 % % % % % % % Corr Coeff R Correltion Coefficient Accurcy Accurcy is the mesure of exctness of n nlyticl method or closeness of greement between the mesured vlue nd the vlue tht is ccepted either s true vlue, conventionl or n ccepted reference. It is generlly mesured s the percentge of nlyte recovered through ssy by spiking smples in blind study. The ccurcy of the method ws determined by nlyzing smples of plcebo spiked with Olmesrtn Medoxomil, Hydrochlorothizide nd Amlodipine t concentrtions representing the levels listed bove. The percent recovery results re summrized on Tble 9, 9b nd 9c. Tble 9 Accurcy -Olmesrtn Olmesrtn % Smple Amount dded Amount found Concentrtion % Recovery (mg/smple preprtion) (mg/smple preprtion) 70 % % Men % Recovery % RSD % % Men % Recovery % RSD % % Men % Recovery % RSD 1.07 Reltive Stndrd Devition Tble 9b Accurcy- Hydrochlorothizide Hydrochlorothizide % Smple Amount dded Amount found Concentrtion % Recovery (mg/smple preprtion) (mg/smple preprtion) 70 % % Men % Recovery % RSD % % Men % Recovery % RSD % % Men % Recovery % RSD 0.70 Reltive Stndrd Devition 189
9 Tble 9c Accurcy- Amlodipine Amlodipine % Smple Amount dded Amount found Concentrtion % Recovery (mg/smple preprtion) (mg/smple preprtion) 70 % % Men % Recovery % RSD % % Men % Recovery % RSD % % Men % Recovery % RSD 0.36 Reltive Stndrd Devition Robustness Robustness is the cpcity of method to remin unffected by smll deliberte vritions in method prmeters. The robustness of method is evluted by vrying prmeters such s percent orgnic solvent, ph, temperture, wvelength, mobile phse flow rte nd determining the effect on the results of the method. Deliberte vritions in the method prmeters were mde in order to demonstrte the robustness of the method. Results re presented in Tble: 10-10i, Fig. 2D. Tble 10 Robustness-Wvelength-Olmesrtn Wve length Theoreticl plte Tiling fctor Absolute difference 235 nm nm nm Tble 10b Robustness-Wvelength-Hydrochlorothizide Wve length Theoreticl plte Tiling fctor Absolute difference 235 nm nm nm Tble 10c Robustness-Wvelength-Amlodipine Wve length Theoreticl plte Tiling fctor Absolute difference 235 nm nm nm Tble 10d Robustness-Flow-Olmesrtn Wve length Theoreticl plte Tiling fctor Absolute difference 235 nm nm nm
10 Tble 10e Robustness-Flow-Hydrochlorothizide Wve length Theoreticl plte Tiling fctor Absolute difference 235 nm nm nm Tble 10f Robustness-Flow-Amlodipine Wve length Theoreticl plte Tiling fctor Absolute difference 235 nm nm nm Tble 10g Robustness-Column Temperture-Olmesrtn Wve length Theoreticl plte Tiling fctor Absolute difference 235 nm nm nm Tble 10h Robustness-Column Temperture-Hydrochlorothizide Wve length Theoreticl plte Tiling fctor Absolute difference 235 nm nm nm Tble 10i Robustness-Column Temperture-Amlodipine Wve length Theoreticl plte Tiling fctor Absolute difference 235 nm nm nm Stbility of stndrd nd smple solution The stbility of the stndrd nd smple were determined by mking series of stndrd nd smple solutions over period of bout 16 h. Percentge Recovery nlysis ws performed on the pek res of Olmesrtn, Hydrochlorothizide nd Amlodipine respectively. Tble: 11-11b. Tble 11 Stndrd solution stbility t room temperture Intervl Olmesrtn Hydrochlorothizide Amlodipine Are % Recovery Are % Recovery Are % Recovery Initil th h th h th h th h Tiling Fctor Theoriticl pltes %RSD
11 Tble 11b Smple solution stbility t room temperture Intervl Olmesrtn Hydrochlorothizide Amlodipine Are % Recovery Are % Recovery Are % Recovery Initil th h th h th h th h Tiling Fctor Theoriticl pltes %RSD Fig.1 : [A] Buffer: Acetonitrile: Methnol composition t 70:10:30 [B] Buffer: Acetonitrile: Methnol composition t 60:20:20 [C] Buffer: Acetonitrile: Methnol composition t 50:40:10 [D] Buffer: Acetonitrile composition t 87:13. Fig.2: [A] Blnk smple [B] Olmesrtn, Hydrochlorothizide, Amlodipine (Stndrd) [C] Linerity for Olmesrtn, Hydrochlorothizide, Amlodipine (Reference stock solution) [D] Olmesrtn, Hydrochlorothizide, Amlodipine (Robustness flow (+) for smple). 192
12 Fig.3: Chromtogrms obtined during different stress conditions [A] Acid stressed for std: Purity curve of Olmesrtn [B] Acid stressed for smple: Purity curve of Olmesrtn [C] Alkli stressed for std [D] Alkli stressed for std : Purity curve of hydrochlorothizide [E] Alkli stressed for std: Purity curve of Olmesrtn [F] Oxidtive stress for smple [G] Oxidtive stress for smple: Purity curve of Olmesrtn [H] Photo stressed for smple [I] Photo stressed for std: Purity curve of hydrochlorothizide [J] UV stressed for std [K] UV stressed for smple : Purity curve of hydrochlorothizide [L] Het stressed for smple: Purity curve of Olmesrtn. 193
13 This Reverse Phse HPLC is commonly used for the quntittive estimtion of drugs from their formultion s well s from the biologicl fluids. Ese of performnce specificity nd nlysis of smple re the importnt fetures of this technique. In the present work different proportion of orgnic solvents were tried for selection nd optimiztion of mobile phse. Sincere ttempt ws mde to increse the difference in retention time by incresing the proportion of Acetonitrile but decresing the volume of buffer nd methnol some extend resulted in the brodening of peks. The mobile phse tht found to be most suitble ws Buffer: Acetonitrile (87:13 v/v) ph 7.0 with sodium hydroxide. This mobile phse composition gve good resolution nd optimum retention time with pproprite tiling fctor (< 2). The detection wve length ws selected s nm where the peks height of the drugs ws cceptble. Finlly the vlidtion of HPLC method ws performed s per ICH guidelines. Fig.4: Linerity grph for Olmesrtn Medoxomil. Fig.5: Linerity grph for Hydrochlorothizide Fig.6: Linerity grph for Amlodipine 194
14 CONCLUSION In conclusion successfully robust HPLC nlyticl method ws developed for the ssy of Olmesrtn Medoxomil, Hydrochlorothizide nd Amlodipine Besylte with respect to dosge form nd method of nlysis. This method is stbility indicting method nd therefore cn be pplied to the formultion for nlysis of the smple for relese nd stbility purpose. Similrly the interference of known impurities ws studied during the development stge. The performnce chrcteristics studied for the optimiztion of the method were Specificity, Precision, Accurcy, Robustness nd Linerity. Acknowledgements The uthors sincerely thnks VIT University Vellore, Tmilndu, Indi for providing ll the necessry fcilities required for crrying out this reserch work. The uthors lso thnk Micro Lbs, Hosur, Krntk for providing HPLC nlysis. REFERENCES [1] S. Gorog, Identifiction nd Determintion of Impurities in Drugs. Elsevier, 2000, P [2] R. Ngeswr Ro, V. Ngrju, J Phrm Biomed Anl., 2003, 33, [3] S. Gorog, Trends Anl Chem., 2006, 25(8), [4] P.D. Hmrpurkr, K.K. Gdpyle, Interntionl Journl of Applied Science nd Engineering., 2013, 11, [5] P.S. Ptil, H.N. More, S.A. Pishwikr, Interntionl Journl of Phrmcy nd Phrmceuticl sciences., 2011, 3, [6] K.H. Vchhni, S.A. Ptel, Journl of Applied Phrmceuticl Science., 2011, 01, [7] S.K. Sinh, P.K. Shrivstv, S.K. Shrivstv, Asin Pcific Journl of Tropicl Biomedicine., 2013, S312- S315. [8] S.H. Kumr, V. Shu, R. Shu, N. Sengr, S. Kulkerni S. Interntionl Journl of Reserch in Phrmcy nd Science., 2011, 1, [9] A.R. Chbukswr, B.S. Kuchekr, S.C. Jgdle, D.M. Mehetre, A.S. More, P.D. Lokhnde. Archives of Applied Science Reserch., 2010, 2, [10] K.K. Chitny, D.G. Snkr, D.S. Isrel, Journl of Globl Trends in Phrmceuticl Sciences., 2013, 4, [11] N. Jin, R. Jin, J. Bnweer, D.K. Jin, Interntionl Journl of Current Phrmceuticl Reserch., 2010, 2, [12] V. Vichre, V. Tmbe, V. Kshikr, S.N. Dhole, Int J Chem Res., 2011, 2, [13] R.A. Mhske, D.J. Grole, A.A. Mhske, S. Shsrbudhe, Interntionl Journl of Phrmceuticl Sciences nd Reserch., 2011, 3, [14] S. Shoo, P.K. Pnd, S.K. Mishr, Int.J.Phrm& Ind.Res., 2011, 3, [15] N.M. Ro, D.G. Snkr, Interntionl Journl of Phrmcy nd Phrmceuticl Science Reserch., 2011, 1, 1-5. [16] Z. Ying, L. Jie, J. Xiomeng, L. Mingzhou, L. Huqun, X. Zhengjing, F. Yue, L. Weiyong, Journl of Phrmceuticl nd Biomedicl Anlysis, 2013, 83, [17] B.G. Chudhri, N.M. Ptel, R.S. Meht, S.L. Bldni, T.R. Gndhi, Indin Drugs., 2006, 43, [18] N.R. Pilli, J.K. Inmdugu, R. Mullngi, V.K. Krr, J.R. Vidy, J.V. Ro, Biomed.Chromtogr, 2011, 26, [19] N. Doshi, A. Sheth, C.N. Ptel, Interntionl Reserch Journl of Phrmcy., 2012, 3, [20] B.N. Nlluri, B.V. Nik, B. Sunndn, K. Sushmith, Journl of Chemicl nd Phrmceuticl Reserch., 2013, 5, [21] K.K. Kumr, C.K. Ro, G. Mdhusudn, K. Mukknti, Americn Journl of Anlyticl Chemistry., 2012, 3, [22] Code Q2A Text on Vlidtion of Anlyticl Procedure Methodology Step-3 Consensus Guideline, ICH Hrmonised Triprtite Guideline, [23] Code Q2B Text on Vlidtion of Anlyticl Procedure Methodology Step-3 Consensus Guideline, ICH Hrmonised Triprtite Guideline, [24] N.D. Rj, S. Anbzhgn, K.A. Bbu, S.N. Bbu, C.N. Bhimndhuni, Interntionl Current Phrmceuticl Journl, 2012, 1,
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Development of an Assay for Besylate in Amlodipine Besylate by IC and a Second Assay to Simultaneously Determine Amlodipine and Besylate by HPLC
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