Reverse Phase High Performance Liquid Chromatography method for determination of Lercanidipine hydrochloride in bulk and tablet dosage form
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1 Research Article ISSN: M.V.Kumudhavalli et al. / Journal of Pharmacy Research 2014,8(11), Available online through Reverse Phase High Performance Liquid Chromatography method for determination of Lercanidipine hydrochloride in bulk and tablet dosage form M.V. Kumudhavalli*,Venkatesh. P, B. Jaykar, Palanisamy. P Department of Pharmaceutical Anaylsis, Vinayaka Mission s College of Pharmacy, Vinayaka Missions University, Yercaud Main Road, Kondappanickenpatty, Salem, Tamil Nadu, India Received on: ; Revised on: ; Accepted on: ABSTRACT A simple, sensitive and rapid reverse phase performance liquid chromatography (RP-HPLC) method was development for the determination of Lercanidipine hydrochloride. The HPLC analysis was performed on the Phenomenex Luna C 18 (250 mm 4.60 mm), 5µm particle size in isocratic mode, at 25 0 C temperature using a mobile phase consisting of methanol: water (95:5 v/v), at a flow rate of. The detection was carried out at 237 nm. The average retention time for LCD was found to be 5.38 min. Linearity was observed in the concentration range 4-28 µg/ml (r 2 = 0.998).The method has been successively applied for the determination of LCD in tablets. There was no interference from the excipients commonly present in tablets. The drug content was found to be %. Accuracy of the method was checked by recovery experiments at three different levels 80 %, 100 % and 120 %. The mean percentage recovery was found to be within the limits of acceptance criteria with average recovery in the range %. The % R.S.D. below 2.0 proved accuracy of proposed method. The results did not show any statistical difference between operators and they suggesting the methods developed was rugged. System suitability tests are an integral part of chromatographic methods. To ascertain its effectiveness, system suitability tests were carried out on freshly prepared stock solution. The method used for the above developed method is precise, accurate, simple and easy for routine analysis. KEYWORDS:Lercanidipine hydrochloride, Linearity and RP-HPLC INTRODUCTION HPLC is a type of liquid chromatography that employs a liquid mobile phase and a very finely divided stationary phase. The technique of high performance liquid chromatography is so called because of its improved performance when compared to column chromatography. Advances in column technology, high-pressure pumping system and sensitive detectors have transformed liquid column chromatography into high speed, efficient, accurate and highly resolved method of separation 1-2. Lercanidipine hydrochloride, a dihydropyridine calcium channel blocker used in the treatment of hypertension. Lercanidipine hydrochloride is available in the tablet dosage form. In literature, very few methods are reported for determination of lercanidipine hydrochloride in pharmaceutical formulations. Therefore, it was thought *Corresponding author. R.Margret Chandira, M. Pharmacy.,Ph.D., Department of Pharmaceutics, Vinayaka Mission s College of Pharmacy, Yercaud Main Road, Kondappanaickanpatty (Post), Salem (D.T),India meaningful. To develop simple, rapid, specific and sensitive RP-HPLC method for the determination of Lercanidipine hydrochloride in tablet dosage form for a routine analysis. To validate the method according to ICH guidelines. MATERILA AND METHODS: Experimental Work Chemicals Lercanidipine hydrochloride 3-5 was gifted by Central Drug Research Institute, (Lucknow). Acetonitrile, triethylamine, methanol, ortho-phosphoric acid, potassium dihydrogen phosphate, ammonium acetate, and tripple distil water were procured from Merck Laboratories. selection of wavelength: Appropriate volume 1.0 ml of standard stock LCD was transferred to 10 ml amber glass volumetric flask and the volume was adjusted to the mark with same solvent i.e 10 µg/ml. The solution was scanned in the UV range nm. The spectrum of LCD was recorded.
2 M.V.Kumudhavalli et al. / Journal of Pharmacy Research 2014,8(11), instrument and washed with Double Distilled water for. Then run the mobile phase for for column saturation. Trial- 1 Table. No :1 Chromatographic conditions for trial-1 Mobile Phase Methanol (100%) 1.0ml/min Temperature Ambient Fig. No. 1 : U.V Spectra of LCD Observation : The drug was shown? max at 237 nm Conclusion : Hence this wavelength was selected for RP-HPLC method analysis. Method Development for the Assay of Lercanidipine Hcl by Reverse Phase HPLC Preparation of solutions Preparation of Mobile Phase: Mobile phases such as methanol, water and varying proportions of methanol: water were tried to achieve separation of LCD. A mobile phase consist of methanol: water in (100%, 80:20, 90:10, 95:5 v/v respectively) was selected to achieve symmetrical peak and sensitivity. The effects of flow rates in the range ml/min were examined. Preparation of standard solution: Standard stock solution was prepared by dissolving 10 mg of LCD in 100 ml methanol that gives concentration 100µg/ml. Fig. No. 1: Chromatogram for Chromatographic condition 1 Observation : No Peak was observed. Trial- 2 Table. No :2 Chromatographic conditions for trial-2 Mobile Phase Methanol: Water(80:20) 1.0ml/min Temperature Ambient Preparation of sample solution: Accurately weighed quantity 10 mg of LCD was transferred to 100 ml calibrated volumetric flask, dissolved in methanol and the volume was adjusted to the mark. The solution was further diluted to get concentration of 10 µg/ml, subjected to analyses and amount of LCD was determined. Initialization of the Instrument First, the column was placed on the instrument and switch on the Fig. No. 2 : Chromatogram for Chromatographic condition
3 Observation : No peak was observed. Trial- 3 M.V.Kumudhavalli et al. / Journal of Pharmacy Research 2014,8(11), Table. No : 3 Chromatographic conditions for trial-3 Mobile Phase Methanol: Water(90:10) 1.5ml/min Temperature Ambient Observation : The retention time of Lercanidipine was 5.38 min Conclusion : Theoretical plate value was observed as A single peak was eluted, and the peak symmetry was good. Hence this condition was selected for validation process. Validation of Developed Method The developed method was validated according to ICH guidelines. The mobile phase was prepared and arranged all parameter as per the above optimized method. Validation System suitability 2. Precision 3. Accuracy (recovery) 4. Linearity of test method 5. Robustness of test method RESULTS AND DISCUSSION Validation data 1. System suitability: Table. No: 5 Result of system suitability parameters for Lercanidipine Fig. No. 3 :Chromatogram for Chromatographic condition 3 Observation : Broad peak was observed. Optimized Method Table. No :4 Chromatographic conditions for the optimized method. System RT AUC No. of Tailing suitability theoretical factor Parameter plates Rep Rep Rep Mean S.D % R.S.D Chromatogram of System Suitability Mobile Phase Methanol: Water(95:5) Temperature Ambient Fig. No. 5 Chromatogram of System Suitability of Lercanidipine HCL Observation : Retention time was observed at 5.37 Result : Standard solution Lercanidipine HCL was determined under proposed condition chromatogram indicating satisfactory retention time and area. Fig. No. 4 : Chromatogram for optimized Chromatographic condition 4
4 2. Precision M.V.Kumudhavalli et al. / Journal of Pharmacy Research 2014,8(11), Observation: Retention time was observed at 5.41 Table. No: 6 Data for intra-day Conc. AUC MEAN S.D %RSD (µg/ml) REP-1 REP-2 REP Table. No: 7 Data for inter-day Conc. AUC MEAN S.D %RSD (µg/ml) REP-1 REP-2 REP Chromatograms of Precision Fig. No. 8 Chromatogram of Lercanidipine HCL at 12 µg/ml concentration: Observation : Retention time was observed at 5.33 Fig. No. 6 Chromatogram of Repeatability of Lercanidipine HCL Observation :Precision %RSD was found to be 1.3%, 0.8% and 0.1 for Repeatability, Analyst 1 and Analyst 2 respectively. This indicates that the method is precise. Intra Day Precision Fig. No. 9 Chromatogram of Lercanidipine HCL at 16 µg/ml concentration Observation: Retention time was observed at Intra Day Precision Fig. No. 10 Chromatogram of Lercanidipine HCL at 8 µg/ml concentration Fig. No. 7 Chromatogram of Lercanidipine HCL at 8µg/ml concentration
5 M.V.Kumudhavalli et al. / Journal of Pharmacy Research 2014,8(11), Observation : Retention time was observed at Chromatograms of Accuracy Fig. No. 11 Chromatogram of Lercanidipine HCL at 12 µg/ml concentration Observation: Retention time was observed at Fig. No. 13 Chromatogram of Accuracy 80 % Lercanidipine HCL Sample Observation: Retention time was observed at Fig. No. 12 Chromatogram of Lercanidipine HCL at 16 µg/ml concentration Observation: Retention time was observed at Fig. No. 14 Chromatogram of Accuracy 100% Lercanidipine HCL Sample Observation: Retention time was observed at Result:The R. S. D of Lercanidipine HCL was found to be presence with in the limits (<2%). 3. Accuracy Table. No: 8 Accuracy of proposed HPLC method %Concentration % Recovery Lercanidipine MEAN S.D. 0.5 % R.S.D Fig. No. 15 Chromatogram of Accuracy 120% Lercanidipine HCL Sample
6 M.V.Kumudhavalli et al. / Journal of Pharmacy Research 2014,8(11), Observation: Retention time was observed at Result:The S. D and R. S. D of Lercanidipine HCL were found to be 0.5 and Linearity Table. No: 9 Linearity and calibration data of Lercanidipine Conc.(µg/ml) Area under curve(auc) replicates Mean Rep-I Rep-II Rep-III r Slop Intercept Table. No: 12 Chromatogram of Lercanidipine HCL at flow rate at 0.9 ml/min: Mobile Phase Methanol: Water(95:5) Temperature Ambient Fig. No. 17 Chromatogram of Lercanidipine HCL e at flow rate at 0.9 ml/min Observation: Retention time was observed at 5.99 Table. No: 13 Chromatogram of Lercanidipine HCL at flow rate at 1.1 ml/min: Fig. No. 16 Linearity Graph of Lercanidipine Hcl 5. Robustness Data variability: Table. No: 10 Effect of variation in the flow rate of (0.9 ml/min) for Lercanidipine Mobile Phase Methanol: Water(95:5) Temperature Ambient Area Retention time Rep Rep Lercanidipine Rep Mean S.D % RSD Table. No: 11 Effect of variation in the flow rate of (1.1 ml/min) for Lercanidipine Area Retention time Rep Rep Lercanidipine Rep Mean S.D % RSD Fig. No. 18 Chromatogram of Lercanidipine HCL at flow rate at 1.1 ml/min
7 M.V.Kumudhavalli et al. / Journal of Pharmacy Research 2014,8(11), Observation : Retention time was observed at 5.10 Mobile phase variability: Table. No: 14 Data for mobile phase Methanol: water (94:06): Area Retention time Rep Lercanidipine Rep Rep Mean S.D % RSD Table. No: 15 Data for mobile phase Methanol: water (96:04): Lercanidipine Area Retention time Rep Rep Rep Mean S.D % RSD Table. No: 16 Chromatogram of Lercanidipine HCL in Methanol: Water (94:06) mobile phase Mobile Phase Methanol: Water(94:06) Temperature Ambient Fig. No. 20 Chromatogram of LercanidipineHCL in Methanol: Water (96:04) mobile phase Observation: Retention time was observed at 5.12 Observation: The % RSD was found within the limits. The all-individual assays of tablets should be within 98% to 102%. Result: Comparison of the results obtained on two different systems and s, showed that the assay test method were rugged for system-to-system variability, mobile phase and flow rate variability s. Table. No: 18 Validation Protocol Summary S.N Validation parameters Lercanidipine Fig. No. 19 Chromatogram of Lercanidipine HCL in Methanol: Water (94:06) mobile phase Observation: Retention time was observed at 6.05 Table. No: 17 Chromatogram of Lercanidipine HCL in Methanol: Water (96:04) mobile phase: Mobile Phase Methanol: Water(96:04) Temperature Ambient 1 Linearity (r 2 ) Precision (%RSD) Intra- Day 8ppm ppm ppm Inter-Day 8ppm ppm ppm Accuracy by recovery study (% RSD found) Robustness(%RSD) a) of (0.9 ml/min) b) of (1.1 ml/min) c) Mobile phase Methanol: water (94:06) d) Mobile phase Methanol: water (94:06) SUMMARY AND CONCLUSION Lercanidipine hydrochloride (LCD), is dihydropyridine calcium and act a channel blocker which is used in the treatment of hypertension. The UV Spectra of LCD show maximum absorbance at 237nm in methanol. Hence the max was selected for further RPHPCL method development. RP-HPLC method has been developed for estimation of LCD in bulk and tablet formulation. The HPLC analysis was performed on the Phenomenex Luna C 18 (250 mm 4.60 mm), 5µm particle size in isocratic mode, at 25 0 C temperature using a mobile phase consisting of methanol: water (95:5 v/v), at a flow rate of. The detection was carried out at 237 nm. The average retention time for LCD was found to be 5.38 min.
8 M.V.Kumudhavalli et al. / Journal of Pharmacy Research 2014,8(11), Linearity was observed in the concentration range 4-28 µg/ml (r 2 = 0.998).The method has been successively applied for the determination of LCD in tablets. There was no interference from the excipients commonly present in tablets. The drug content was found to be %. Accuracy of the method was checked by recovery experiments at three different levels 80 %, 100 % and 120 %. The mean percentage recovery was found to be within the limits of acceptance criteria with average recovery in the range %. The % R.S.D. below 2.0 proved accuracy of proposed method. The results did not show any statistical difference between operators and they suggesting the methods developed was rugged. System suitability tests are an integral part of chromatographic methods. They are used to verify the reproducibility of the chromatographic system. To ascertain its effectiveness, system suitability tests were carried out on freshly prepared stock solution. From this present study it is clear that the above developed method is precise, accurate, simple and easy for routine analysis. ACKNOWLEDGEMENTS Authors are thankful to Prof (Dr.).B.Jaykar, Principal Vinayaka Mission s College of Pharmacy, Salem, Tamil nadu and providing all the facilities for this research project. REFERENCES 1. Snyder LR, Kirkland JJ and Glajch LJ (1997) Practical HPLC Method Development, 2 nd edn, John Wiley and Sons, INC, pp Sethi PD (1996) HPTLC: Quantitative Analysis of Pharmaceutical Formulation, CBS Publications, New Delhi, pp Maryadele JO Neil (2006) The Merck Index, Eds. In, 14th edn, Published by Merck and Co, White House Station, NJ, USA, pp Sweetman SC, Martindale (2007) The Extra Pharmacopoeia, The complete Drug Reference, 35 th edn,pharmaceutical Press, London, 2007, pp British Pharmacopoeia, Vol. I, HMSO, Cambridge, International edn, 2005, pp Reviewer Guidance, Validation of Chromatographic Methods (CDER), Nov ICH Guidelines Q2A, Validation of Analytical Procedures: Definition and terminology (CPMP III/5626/94) March (1995) Geneva, Switzerland. 8. ICH Guidelines Q2B, Validation of Analytical Procedures: Methodology (CPMP/ICH/281/95) November (1996) Geneva, Switzerland. 9. Indian Pharmacopoeia, Vol-I, Govt. of India, The Controller of Publication, New Delhi, 1996, pp-72. Source of support: Nil, Conflict of interest: None Declared
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