Simultaneous Quantitative Analysis of Olmesartan Medoxomil and Amlodipine Besylate in Plasma by High-performance Liquid Chromatography Technique
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1 Phrmceuticl Anlysis Simultneous Quntittive Anlysis of Olmesrtn Medoxomil nd Amlodipine Besylte in Plsm by High-performnce Liquid Chromtogrphy Technique Shh SK, Asnni AJ 1, Kwde DP 1, Dngre SC 2, Aror SK 3, Yende SR 4 Deprtment of Phrmceuticl Chemistry, Smt. Kishoriti Bhoyr College of Phrmcy, Ngpur, 1 J. L. Chturvedi College of Phrmcy, MIDC, Ngpur, 2 Shrd Pwr College of Phrmcy, Wndongri, Ngpur, 3 Deprtment of Phrmceuticl Science, Rshtrsnt Tukdoji Mhrj Ngpur University, Amrvti Rod, Ngpur, 4 Gurunnk College of Phrmcy, Ngpur, Mhrshtr, Indi Address for correspondence: Prof. Spn Kmleshkumr Shh; E-mil: shh.spn@rediffmil.com ABSTRACT -5 phrmcodynmic study of OLM nd AM in combined dosge form. Key words: Amlodipine besylte, high-performnce liquid chromtogrphy, olmesrtn medoxomil, plsm INTRODUCTION methyl-1, 4-dihydropyridine [Figure 1], is potent clcium Access this rticle online Quick Response Code: Website: DOI: / chnnel blocker used in tretment of hypertension nd ngin pectoris. [1] Amlodipine is well bsorbed following orl dministrtion with pek blood concentrtions occurring fter 6 12 h. The biovilbility is bout 60 65%. It hs prolonged terminl elimintion hlf-life of h, nd stedy-stte plsm concentrtions re not chieved until fter 7 8 dys of dministrtion. Amlodipine s unchnged drug. [2,3] Severl nlyticl methods for reported; such s cpillry gs chromtogrphy (GC) with electron cpture detection, [4,5] GC with electron-impct 88 Journl of Young Phrmcists Vol 4 / No 2
2 Figure 1: mss spectrometry (EI-MS), [6,7] nd high-performnce liquid chromtogrphy (HPLC). [8-12] Severl HPLC procedures hve been lso reported for the nlyses of mlodipine bsed on MS MS detection by using tndem mss spectrometry. [13-16] y-1-methylethyl)-2-propyl-1-{4-[2-(tetrzol-5-yl)-phenyl] potent nd selective ngiotensin AT1 receptor blocker [17] which hs been pproved for the tretment of hypertension rpidly by n endogenous esterse to relese the ctive metbolite olmesrtn. [18] [19,20] As per literture survey, no nlyticl methods hve been reported for the simultneous quntittive nlysis of ws to develop rpid, simple, ccurte, precise, sensitive, nd reproducible HPLC method, which cn be utilized in phrmcokinetic reserch. Chemicls nd regents EXPERIMENTAL (AM) were kindly supplied by Ajnt Phrm Limited (Mumbi). All chemicls nd regents were HPLC grde, methnol nd cetonitrile, mmonium cette, ethylene dimine tetr cetic cid (EDTA) (Merck, Indi). Instruments nd chromtogrphic conditions A Shimdzu HPLC system (Jpn) employed consisted of model LC-20 AT pump, model Rheodyne 7125 injector, performed on n nlyticl mm Eurospher C18 (5 m, prticle size) column. The wvelength Figure 2: cetonitrile:0.05 M mmonium cette buffer: 0.1 ml 1.0 ml/min. The mobile phse ws prepred dily nd Preprtion of stndrd stock solution The stndrd working concentrtion of OLM (10 g/ml) nd AM (10 g/ml) were prepred in the mobile phse. Biologicl smples The prepred suspension contining AM (0.4 mg/ml) nd OLM (1.6 mg/ml) in 0.2% tween 80 were dministered orlly to Wistr rts ( g) t dose of 2 mg/kg (AM) nd 8 mg/kg (OLM) body weight fter overnight fsting. The into nticogulnt-treted polypropylene tubes from 0 to 12 h fter drug dministrtion. Blood smples collected were centrifuged immeditely to seprte the plsm. The plsm then processed for drug recovery vi Smples preprtion (extrction procedure) To 1000 L of plsm smples in borosilicte glss tube centrifuged t 10,500 rpm for 10 min. The upper orgnic lyer ws trnsferred to glss continer nd evported inside vcuum oven t 40 C. The dry residue ws sonicted well for 10 min, nd 20 L of this solution ws injected into liquid chromtogrphy. Stbility chnge in concentrtion of n nlyte in plsm under Journl of Young Phrmcists Vol 4 / No 2 89
3 plsm ws investigted by dding known mount of drug to blnk plsm smples to give concentrtions of 10, 200, nd 500 ng/ml. These were stored t -20 C nd liquots tken for 1 nd 4 weeks for nlysis. Clibrtion curve Linerity of instrument stndrd response ws determined for ech compound with different concentrtion 2500 ng/ml for AM nd 8 to 10,000 ng/ml for OLM, with drug rtio mintining constnt 1:4). The clibrtion curves were constructed by plotting individul nlyte pek re rtio s compred to the corresponding concentrtion nd were constructed by the weighted regression method (1/x) of the pek re of OLM or AM vs. ctul concentrtions. Method development RESULTS AND DISCUSSION Considering the efficiency of HPLC, n ttempt hs been mde to develop simple, ccurte, precise, rpid, nd economic method for the simultneous estimtion of OLM nd AM in the biologicl method. Thus, the nd AM. The dvntges lie in the simplicity of smple preprtion nd the low costs of regents used. For the HPLC method development mobile phse consists of cetonitrile:0.05 M mmonium cette buffer:0.1 ml chromtogrphic conditions described, OLM nd AM peks were well resolved. Endogenous plsm components did not give ny interfering peks. The verge retention times of OLM nd AM were 3.1 nd 5.0 min, respectively. Chromtogrm obtined fter drug dministrtion of OLM nd AM in Wistr rts for blnk plsm nd plsm smples collected fter 2.5, 6 nd 10 h re shown in Figure 3. Clibrtion curve The clibrtion curve for the determintion of OLM nd AM in plsm ws liner over the rnge nd 8 10,000 ng/ml AM nd OLM. The linerity of this curve, the intercept ws not sttisticlly different from zero. equl to or better thn The reltive stndrd devition (RSD) vlues of the slope were equl to or better thn 5%. For ech point of clibrtion stndrds, the concentrtions were reclculted from the eqution of the liner regression curves. Recovery study The reltive nlyticl recovery for plsm t three different concentrtions of OLM nd AM ws determined. Known mounts of drug were dded to drug-free plsm in concentrtions rnging from to 10 g/ml. The verge recovery ws in between 50% nd 90% for liner concentrtion rnge. The results re given in Tbles 1 nd 2 for the intrdy nd interdy study. Stbility study In the short-term stbility studies, precision nd ccurcy for the nlyte ws <15% nd <8% for OLM, <10% nd 8% for AM, respectively. In the long-term stbility studies, precision nd ccurcy for the nlyte ws <7% nd <15% % ±SD CV Tble 1: Recovery studies for olmesrtn medoxomil nd mlodipine besylte (intrdy) (RP-HPLC) Smple Drug Concentrtion Pek re Men pek dded (μg ml) of stndrd re smple Recovery 1 AM OLM AM OLM AM OLM AM OLM AM OLM AM OLM AM OLM OLM: Olmesrtn medoxomil; AM: Amlodipine besylte; Results re men of three smples; RP-HPLC: Reversed phse HPLC; SD: Stndrd devition 90 Journl of Young Phrmcists Vol 4 / No 2
4 Figure 3: for OLM, <12% nd 18% for AM, respectively. The stbility tests indicted tht OLM nd AM re stble in rt plsm over the period of 1 month. In ddition, the stock solution remins stble for durtion of 1 month when OLM nd AM. The results re given in Tbles 3 nd 4 for OLM nd AM, respectively. Limit of detection The limit of detection (LOD) ws defined, s the mlodipine concentrtion tht produced signl-to-noise rtio greter thn 3. The LOD in plsm ws 2 ng/ml for AM nd 8 ng/ml bsed upon this criterion. At this level, the RSD ws lower thn 15%. Accurcy nd precision Intrdy nd interdy ssy performed to evlute precision intrdy studies ws between 2 14% nd 3 17% for interdy in rt plsm. The result suggests tht within run for the nlyte met the cceptnce criteri. It does not require tedious derivtiztion or specilized detectors, mking it redily mendble to routine lbortory use. However, this method is sensitive enough for drug monitoring nd other purposes such s phrmcokinetic studies. We ssessed the precision of the method by repeted nlysis of plsm specimens contining known concentrtions of mlodipine nd olmesrtn. is cceptble for the routine mesurement of OLM nd AM. The results for intrdy nd interdy precision nd ccurcy studies re given in Tbles 5 nd 6. Phrmcokinetic study The results of phrmcokinetics nlysis of OLM nd AM re mentioned in Tble 7. The men plsm concentrtion time curve of OLM nd AM shows: Journl of Young Phrmcists Vol 4 / No 2 91
5 Tble 2: Recovery studies for OLM nd AM (interdy) (RP-HPLC) Smple Drug Concentrtion dded (μg/ml) Pek re of stndrd Men pek % ±SD CV re smple Recovery 1 AM OLM AM OLM AM OLM Tble 3: Stbility of OLM in rt plsm (RP-HPLC) Smple concentrtion (ng ml) Concentrtion found (%) ±SD Men concentrtion (%) CV % Devition (inccurcy) Tble 4: Stbility of AM in rt plsm (RP-HPLC) Smple concentrtion (ng ml) Concentrtion found (%) ±SD Men concentrtion (%) CV % Devition (inccurcy) ±SD CV % devition Tble 5: Dt for ccurcy nd precision studies (Intrdy) (RP-HPLC) Smple Drug Concentrtion Pek re Men concentrtion dded (μg ml) of stndrd found 1 AM OLM AM OLM AM OLM AM OLM AM OLM AM OLM AM OLM Results re men of three smples, RP-HPLC: Reversed phse HPLC; AM: Amlodipine besylte; OLM: Olmesrtn medoxomil for OLM nd AM, fter dosing with level of 8 nd 2 mg/kg body weight of OLM nd AM, respectively. The vlue of phrmcokinetic prmeters such s t 1/2, C nd k el obtined revels tht there ws less difference simultneously in single orl dosge form. These results re greement with the previous reports. [19,21,22] Plsm 92 Journl of Young Phrmcists Vol 4 / No 2
6 ±SD CV % Tble 6: Dt for ccurcy nd precision studies (interdy) (RP-HPLC) Smple Drug Concentrtion Pek re Men concentrtion dded (μg/ml) of stndrd found Devition 1 AM OLM AM OLM AM OLM Results re men of three smples, AM: Amlodipine besylte; OLM: Olmesrtn medoxomil; RP-HPLC: Reversed phse HPL Tble 7: Phrmcokinetic prmeters of single orl dministrtion of OLM nd AM in combined dosge form in Wistr rts Prmeters Drug dministered OLM AM t mx (hrs) 6 2 C mx (μg/ml) AUC 0-t (μg/ml/hrs) AUC INF (μg/ml/hrs) t 1/2 (hrs) CL (μl/hr) AUMC LAST (μg/ml*hr 2 ) AUMC INF (μg/ml*hr 2 ) MRT LAST (hrs) MRT INF (hrs) *Results re men of three smples, OLM: Olmesrtn medoxomil; AM: Amlodipine besylte; AUC: Are under curve; CL: Drug clernce; AUMC: Are under the moment curve evlution nd reserch guidelines for bionlyticl method vlidtion. [23] These methods re well-suited for routine ppliction in the qulity control lbortories nd clinicl lbortories becuse of the simplicity, economic, ccurcy, sensitivity, nd reproducibility. The HPLC method is pplicble to phrmcokinetics studies of OLM nd AM in rts. This method cn lso be used to study the mechnism of metbolism of OLM nd AM. [24] ACKNOWLEDGMENTS The uthors re thnkful to Dr. D. R. Chple, Principl, J. L. Chturvedi College of Phrmcy, Ngpur for providing lbortory fcilities. The uthors lso wish to thnk Mr. Anwr Dud, Mnging Director, Zim Lbortories Ltd., Ngpur, for providing fcilities nd environment conducive for n idel REFERENCES Figure 4: dministrtion of OLM nd AM in combined dosge form in Wistr rts is shown in Figure 4. CONCLUSION The HPLC method presented is direct, simple, selective, reproducible, sensitive, nd liner. The procedure ws successfully pplied to the simultneous determintion interference from the dditives nd endogenous substnces. The procedure ws fully vlidted to meet the requirements of the food nd drug dministrtion center for drug 1. Arrowsmith JE, Cmpbell SF, Cross PE, Stubbs JK, Burges RA, Grdiner DG, et l. Long-cting dihydropyridine clcium ntgonists. J Med Chem 1986;29: ;118: Meredith P, Elliott H. Clinicl phrmcokinetics of mlodipine. Clin Phrmcokinet 1992;22: Krol G, Noe A, Yeh S, Remsch K. Gs nd liquid chromtogrphic nlyses J Chromtogr 1984;305: Bereford A, MCre P, Stopher D, Wood B. Anlysis of mlodipine in humn plsm by gs chromtogrphy. J Chromtogr 1987;420: hydrochloride levels by gs chromtogrphy-mss spectrometry. J Chromtogr 1981;223: Murer HH, Arlt JW. Screening procedure for detection of dihydropyridine nlysis procedure for cidic compounds by gs chromtogrphy-mss 8. Shimook K, Swd Y, Ttemtsu H. Anlysis of mlodipine in serum by sensitive high-performnce liquid chromtogrphic method with mperometric detection. J Phrm Biomed Anl 1989;7: Pollen K, Yeung F, Mosher S, Pollk P. Liquid chromtogrphy ssy for mlodipine: Chemicl stbility nd phrmcokinetics in rbbits. J Phrm Biomed Anl 1991;9: Josefsson M, Norlnder B. Coupled-column chromtogrphy on Chirl-AGP phse for determintion of mlodipine enntiomers in humn plsm: An HPLC ssy with electrochemicl detection. J Phrm Biomed Anl 1996;15: Ttr S, Atmc S. Determintion of mlodipine in humn plsm by Journl of Young Phrmcists Vol 4 / No 2 93
7 et l.: Olmesrtn medoxomil nd mlodipine besylte in plsm J Chromtogr B 2001;758: Bhrmi G, Mirzeei S. Simple nd rpid HPLC method for determintion phrmcokinetic studies. J Phrm Biomed Anl 2004;36: Zhong D, Chen X, Gu J, Li X, Guo J. Applictions of liquid chromtogrphy tndem mss spectrometry in drug nd biomedicl nlyses. Clin Chim Act 2001;313: Chen L, Zhou L, Shen M, M A. Simultneous determintion nd phrmcokinetic study of metformin nd rosiglitzone in humn plsm by HPLC ESI-MS. J Chromtogr Sci 2011;49: Streel B, Line C, Zimmer C, Sibenler R, Ceccto A. Enntiomeric determintion of mlodipine in humn plsm by liquid chromtogrphy coupled to tndem mss spectrometry. J Biochem Biophys Methods 2002;54: Rojnsthien N, Teekchunhten S, Jkob K, Gupp M, Arnold P, Chichn N, et l. Bioequivlence study of generic mlodipine in helthy Thi mle volunteers. Int J Clin Phrmcol Ther 2004;42: Kog K, Ymgishi S, Tkeuchi M, Ingki Y, Amno S, Okmoto T, et l. CS-866, new ngiotensin II type 1 receptor ntgonist, meliortes glomerulr nionic site loss nd prevents progression of dibetic nephropthy in Otsuk Long-Evns Tokushim ftty rts. Mol Med 2002;8: Schwocho L, Msonson H. Phrmcokinetics of CS-866, new ngiotensin II receptor blocker, in helthy subjects. J Clin Phrmcol 2001;41: Nkmur H, Inoue T, Arkw N, Shimizu Y, Yoshige Y, Fujimori I, et l. niml dibetic retinopthy models. Eur J Phrmcol 2005;512: Sellin L, Stegbuer J, Leis P, Rump LC. Adding hydrochlorothizide to olmesrtn dose dependently improves 24-h blood pressure nd response rtes in mild-to-moderte hypertension. J Hypertens 2005;23: ;118: Crvlho M, Oliveir CH, Mendes GD, Sucupir M, Mores ME, De Nucci G. Amlodipine bioequivlence study: Quntifiction by liquid chromtogrphy coupled to tndem mss spectrometry. Biophrm Drug Dispos 2001;22: Administrtion, Guidnce for Industry: Bionlyticl Method vlidtion. 24. Zrghi A, Foroutn SM, Shfti A, Khoddm A. Vlidted HPLC method for determintion of mlodipine in humn plsm nd its ppliction to phrmcokinetic studies. Frmco 2005;60: How to cite this rticle: SK, Yende SR. Simultneous quntittive nlysis of olmesrtn medoxomil nd mlodipine besylte in plsm by high-performnce liquid chromtogrphy technique. J Young Phrmcists 2012;4: Source of Support: Nil, Con ict of Interest: None declred. Author Help: Reference checking fcility The mnuscript system ( llows the uthors to check nd verify the ccurcy nd style of references. The tool checks the references with PubMed s per predefined style. Authors re encourged to use this fcility, before submitting rticles to the journl. The style s well s bibliogrphic elements should be 100% ccurte, to help get the references verified from the system. Even single spelling error or ddition of issue number/month of publiction will led to n error when verifying the reference. Exmple of correct style Shehn P, O lery G, Lee G, Fitzgibbon J. Cystic cervicl metstses: Incidence nd dignosis using fine needle spirtion biopsy. Otolryngol Hed Neck Surg 2002;127: Only the references from journls indexed in PubMed will be checked. Enter ech reference in new line, without seril number. Add up to mximum of 15 references t time. If the reference is correct for its bibliogrphic elements nd punctutions, it will be shown s CORRECT nd link to the correct rticle in PubMed will be given. If ny of the bibliogrphic elements re missing, incorrect or extr (such s issue number), it will be shown s INCORRECT nd link to possible rticles in PubMed will be given. 94 Journl of Young Phrmcists Vol 4 / No 2
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