SIMULTANEOUS ESTIMATION OF NEBIVOLOL HYDROCHLORIDE AND AMLODIPINE BESYLATE IN COMBINED TABLET DOSAGE FORM BY Q-ANALYSIS METHOD Chandan Kumar Giri 1 *, M.S. Kondawar and D.D. Chougule 1 Chandan Kumar 1 Appasaheb Birnale College of Pharmacy, Sangli, Maharashtra, India Email: chandanqa@gmail.com ABSTRACT A simple, accurate, precise, rapid and economical spectrophotometric method for simultaneous estimation of Nebivolol hydrochloride and Amlodipine besylate in combined tablet dosage form has been developed utilizing Q-analysis method or Absorbance ratio method. In this method, absorbance is measured at two wavelengths, one being the iso-absorptive point 268nm (λ1) and the other being λmax of one of the sample components 282nm (λ2). Both the drugs obey Beer s law in the concentration ranges employed for this method. Result of the method was validated statistically as well as by recovery studies. Proposed method for simultaneous estimation of NH and AB in combined sample solutions was found to be simple, accurate and reproducible. Once the equations are determined, analysis required only the measurement of the absorbances of the sample solution at the two wavelengths selected, followed by a few simple calculations. It is a novel method that can be employed for routine analysis in quality control or R & D laboratory. Key Words: Nebivolol hydrochloride, Amlodipine besylate, Spectrophotometric analysis, Q-Analysis method. INTRODUCTION Nebivolol hydrochloride (NH), chemically α, α - [Iminobis (methylene)] bis [6-fluoro-3, 4-dihydro- 2H-1-benzopyran-2-methanol] is a betaadrenergic blocker. Nebivolol selectively blocks the β1- adrenoceptor. Nebivolol reduces heart rate, rate of myocardial contractility and systemic blood pressure while increasing diastolic pause. β-blockers are useful 1
prophylactic agents in stable and unstable types of angina. Nebivolol is preferable in patients with bronchi spasm, diabetes, peripheral vascular disease or Raynaud s phenomenon. The drug is not official in any pharmacopoeia. A survey of literature revealed that there are a number of methods for the individual determination of Nebivolol hydrochloride such as a HPLC- fluorescence method, a UVcolorimetric method, and HPTLC method. Amlodipine besylate is chemically R,S-2-[(2- aminoethoxy) methyl]-4-(2-chloroethyl)-3-ethoxy carbonyl-5-methoxy carbonyl-6-methyl-1,4- dihydropyridine benzene sulphonate used in the treatment of hypertension and congestive heart failure. Literature survey reveals that there are a number of methods for the individual determination of Amlodipine like RP-HPLC, difference spectrophotometry, colorimetry, HPTLC, GC, LC Mass spectrometry and few spectrophotometric methods. There is no such method available for the estimation of Nebivolol hydrochloride and Amlodipine besylate in combined tablet dosage forms. MATERIALS AND METHOD: Instrument: UV/Visible spectrophotometer (JASCO) V-550, Digital balance (SHIMADZU) AUX-220 Amlodipine besylate equivalent to Amlodipine 5 mg and Nebivolol hydrochloride equivalent to Nebivolol 5 mg. Preparation of standard stock solution: Standard stock solutions containing Nebivolol hydrochloride (NH) and Amlodipine besylate (AB) were prepared individually by dissolving 10 mg of NH and 10mg of AB separately in 50ml of methanol. It was then sonicated for 10 minutes and the final volume of both the solutions were made up to 100 ml with methanol to get stock solutions containing 100µg/mL each of NH and AB in two different 100 ml volumetric flasks. NH exhibited λmax at 282 nm and AB showed λmax at 238 and 360 nm. Both of these drugs show isobestic points at 268 nm. Wavelength 282 nm with reference to 268 nm (isobestic point) was selected as the working wavelength, as at this wavelength there is minimum interference of the other drug. Optimization of concentrations of the standards: Concentrations of 10.0 µg/ml of Nebivolol hydrochloride and 10.0 µg/ml of Amlodipine besylate were used as standard 1 and standard 2. The Q values and absorptivities for both the drugs were calculated as follows: Reagents: Methanol of Analytical grade Standard drug: Nebivolol hydrochloride and Amlodipine besylate from Alkem Laboratory, Mumbai. Tablet dosage form: AMLOPRES-NB Tablets from CIPLA, Each uncoated tablet contains Fig. 1: Overlain spectrum of Nebivolol hydrochloride and Amlodipine besylate 2
Regression and optical characteristics of Nebivolol hydrochloride and Amlodipine besylate (Refer Table No: 1) Absoptivity of AB at 282nm QAB= = 0.833 Absorptivity of AB at 268nm Absoptivity of NH at 282nm QNH= =1.4545 Absoptivity of AB at 282nm Absorbance of Mixture at 282nm QM= Absorbance of mixture at 268nm At 268nm the standard equations are as follows: Amlodipine besylate - y = 0.012x + 0.002 Nebivolol hydrochloride- y = 0.011x + 0.001 Amount of each drug was determined using following formulas: QM QNH A CAB = x ------------------ 1 QAB QNH aab QM QAB A CNH = x ------------------ 2 QNH QAB anb Where, CAB = Concentration of AB in gm/100 ml. CNH = Concentration of NH in gm/100 ml. QM = Ratio of absorbance of mixture at λmax and λiso QAB = Ratio of absorptivity of AB at λiso and λmax QNH = Ratio of absorptivity of NH at λiso and λmax A = Absorbance of mixture at isoabsorptive point. aab = E (1%, 1cm) of AB at λiso anb = E (1%, 1cm) of NH at λiso Analysis of tablet formulation: Twenty tablets of Nebivolol hydrochloride and Amlodioine besylate combination were weighed and their average weight was determined. Afterwards, tablets were crushed to powder sample. From the triturate, tablet powder equivalent to 10.0 mg of NH and 10 mg of AB was weighed and transferred to 100 ml volumetric flask and dissolved in 50 ml methanol. The content was kept in ultrasonicator for 20 min. Finally, the volume was made up to the mark with methanol. The solution was filtered through Whatman filter paper No.41. This tablet solution was further diluted to obtain 10 µg/ml for NH and 10 µg/ml for AB. The mixed sample solutions were analyzed to obtain spectra. Absorbance values at 268 nm (isobestic point) and 282 nm were noted. The concentrations of Nebivolol hydrochloride and Amlodipine besylate were calculated from the equation (1) and (2). The results of the analysis of tablet formulation are reported in Table No.3 and data for statistical validation are given in Table No.4. Recovery Studies: Recovery studies were carried out by applying the method to pre-analyzed drug sample in tablet dosage form to which known amount of NH and AB corresponding to 80%, 100% and 120% of label claim was added (standard addition method). The mixed sample solutions were analyzed as per tablet formulation. At each level, three determinations were performed. Results of recovery studies and statistical evaluation are shown in Table No. 5 and 6, respectively 3
RESULTS: Standardization of the method by analysis of powder mixture of known composition (Refer Table No: 2), Analysis of Tablet Formulation (Refer Table No: 3), Statistical validation of Tablet (Refer Table No: 4), Recovery Studies(Refer Table No: 5), Statistical Validation for the Recovery of Tablet Formulation(Refer Table No: 6) DISCUSSION : The proposed method for simultaneous estimation of AB and NH dosage forms were found to be simple, accurate, economical and rapid. The method was validated as per the ICH and USP guidelines. The values of SD or RSD are within the prescribed limit of 2 %, showing high precision of method and recovery was close to 100% for both the drugs. During the linearity stud y it was observed that absorbance values of AB and NB in the marketed formulation were linear in the range of 80% to 120% of test concentration with R2 close to one. CONCLUSION: Proposed method for simultaneous estimation of NH and AB in combined sample solutions was found to be simple, accurate and reproducible. Once the equations are determined, analysis required only the measurement of the absorbances of the sample solution at the two wavelengths selected, followed by a few simple calculations. It is a novel method that can be employed for routine analysis in quality control or R & D laboratory. ACKNOWLEDGEMENTS: The authors are thankful to the Alkem lab, Mumbai for providing gift sample of standard Amlodipine besylate and Nebivlol hydrochloride. The authors also thankful to Cipla pharmaceutical for providing gift sample of Tablet dosage form REFERENCES: 1. ICH Q2A; Guidelines on validation of analytical procedure; Definitions and terminology, Federal Register, 1995, 60, 11260. 2. ICH Q2B; Guidelines on validation of analytical procedure; Methodology, Federal Register, 1996, 60, 27464. 3. PS Tarte*, SP Wate, PB Khedikar, BT Satpure and G Pawnikar Simultaneous Estimation of Nebivolol and Hydrochlorothiazide in Combined Tablet Dosage Form by Multicomponent Mode of Analysis www.ajrconline.org ISSN 0974-4169; AJRC 4. Singh, M. Bakshi, Understanding Analytical Method Validation. Pharma. Times, August, 1999, pg. 15-20. 5. ICH Guidance on Analytical Method Validation, International Convention on Quality for the Pharmaceutical Industry, Toronto, Canada, September, 2002 6. Rao A. L. *, Rajeswari K.R. And Sankar G.G. (2009) Spectrophotometric Method for the Determination of Nebivolol Hydrochloride in Bulk and Pharmaceutical FormulationsISSN: 0973-4945; CODEN ECJHAO E-Journal of Chemistry http://www.e-journals.net 2010, 7(2), 445-448. 7. Dhandapani B. *, Thirumoorthy N. And Prakash D.J. (2009) Development and Validation for the Simultaneous Quantification of Nebivolol Hydrochloride and Hydrochlorothiazide by UV Spectroscopy, RP-HPLC and HPTLC in Tablets, ISSN: 0973-4945; CODEN ECJHAO E-Journal of Chemistry 4
http://www.e-journals.net 2010, 7(2), 341-348 8. Joshi S. J., Karbhari P. A. and Bhoir S. I.( 2009) RP-LC Simultaneous Determination of Nebivolol Hydrochloride and Amlodipine Besilate in Bi-Layer Tablets, Journal of Chromatographia, ISSN0009-5893, Volume 70, 557-561 9. V. Juyal, Chaudhary M., Kumar P., Gnanarajan G., P. K. Yadav (2008) Method development and its validation for simultaneous estimation of Atorvastatin and Amlodipine in combination in tablet dosage form by UV spectroscopy, using multicomponent mode of analysis Journal of Pharmacy Research.01/01/2008; WWW.DOAJ.ORG,IS SN-09746943,2008,volume-1,issue-2,page- 182 10. Birajdar A. S. *, Meyyanathan S. N., and Bhojraj Suresh (2009) Simultaneous Estimation of Nebivolol Hydrochloride and Valsartan and Nebivolol Hydrochloride and Hydrochlorothiazide in Pharmaceutical Formulations by UV Spectrophotometric Methods, Indian J.Pharm. Educ. Res. 44(2), Apr-Jun, 2010 11. SHAH K. V. *, Thumber B. L., Desai T. R. (2009) Simultaneous Estimation of Nebivolol Hydrochloride and Hydrochlorothiazide in Bulk and Tablet Dosage Form by Q-Analysis Method International Journal of Pharmaceutical Science & Research (2009) 1(2) 80-85 12. Patil P. R. *, Rakesh S. U., Dhabale P.N., and Burade K.B.,development and statistical validation of uv spectrophotometric method for estimation of amlodipine besylate in tablet dosage form, ISSN 0975 2633 Arch Pharm Sci & Res Vol 1 No 2 158-161 October 2009 13. Garg G., Saraf S. and Saraf S. (2008) Development and validation of simultaneous estimation of enalapril maleate and amlodipine besylate in combined dosage forms. Trends Applied Sci. Res., 3: 278-284. 14. Kakde R.B. *, Kotak V.H., Barsagade A.G., NK Chaudhary and DL Kale (2008) Spectrophotometric Method for Simultaneous Estimation of Amlodipine Besylate and Bisoprolol Fumarate in Pharmaceutical Preparations, ISSN 0974-3618 www.rjptonline.org, 513 15. Kamble A. Y.; Mahadik M. V.; Khatal L. D.; Dhaneshwar S. R. (2010) Validated HPLC and HPTLC Method for Simultaneous Quantitation of Amlodipine Besylate and Olmesartan Medoxomil in Bulk Drug and Formulation, www.informaworld.com,volume 43, Issue 2 January 2010, pages 251-258 16. Rahman Nafisur and Azmi Syed Najmul Hejaz (2001) Spectrophotometric method for the determination of amlodipine besylate with ninhydrin in drug formulations,www.sciencedirect.com, Volume 56, Issue 10, 1 September 2001, Pages 731-735 17. Vora D. N. * and Kadav A. A. Development and Validation of a Simultaneous HPLC Method for Estimation of Bisoprolol Fumarate and Amlodipine Besylate from Tablets, Indian J Pharm Sci. 2008 Jul Aug; 70(4): 542 546. 5
Tables and Figures: Table 1: Regression and optical characteristics of Nebivolol hydrochloride and Amlodipine besylate. Parameters AB NH λmax in methanol 238 282 Beer s law limit (µg/ml) 2-40 2-80 Correlation coefficient (r) 0.9996 0.9995 Molar absorptivity (lit/mole/cm) 21378.65 53997.32 Slope 0.0376 0.0165 Intercept -0.0047-0.0158 Table 2: Standardization of the method by analysis of powder mixture of known composition: Concentration present (µg/ml) Concentration found (µg/ml) % Estimated AB NH AB NH AB NH 10 10 10.12 9.94 101.2 99.4 10 10 10.08 9.97 100.8 99.7 10 10 10.02 9.98 100.2 99.8 10 10 10.04 9.99 100.4 99.9 10 10 10.10 10.03 101.0 100.3 10 10 10.07 10.06 100.7 100.6 6
Table 3: Analysis of Tablet Formulation Label claim (mg/ tablet) Amount found* (mg/ tablet) Percentage found AB NH AB NH AB NH 5 5 5.05 4.97 101.0 99.4 5 5 5.07 4.98 101.4 99.6 5 5 5.00 5.03 100.0 100.6 5 5 5.05 4.99 101.0 99.8 5 5 5.01 5.00 100.2 100.0 5 5 5.06 5.02 101.2 100.4 Table 4: Statistical validation of Tablet Drug mean Standard deviation Coefficient of variation Standard error Amlodipine 100.80 0.516398 0.512 0.21 Nebivolol 99.96 0.422953 0.423 0.172 Table 5: Recovery Studies Drug mean Standard deviation Coefficient of variation Standard error Amlodipine 100.80 0.516398 0.512 0.21 Nebivolol 99.96 0.422953 0.423 0.172 7
Table 6: Statistical Validation for the Recovery of Tablet Formulation Level of %recovery mean of % recovery Standard deviation Coefficient of variation Standard error DRUG AB NH AB NH AB NH AB NH 80 100.88 99.07 0.273 0.455 0.271 0.460 0.111 0.186 100 100.26 100.2 0.612 0.294 0.611 0.293 0.25 0.12 120 100.6 100.08 0.224 0.199 0.223 0.198 0.0916 0.0812 8