Department of Pharmaceutical Analysis, SRM College of Pharmacy, SRM University, Kattankulathur, Kanchipuram 603203, Tamil Nadu, India.
Received: 04 Aug 2014 Revised and Accepted: 05 Sep 2014
Objective: To develop a new, simple and sensitive visible spectrophotometric method for the estimation of Cilnidipine [CIL] in bulk and tablet dosage forms.
Methods: The visible spectrophotometric method was based on the color reaction of CIL with N-(1-Naphthyl) ethylene diamine dihydrochloride reagent [it is stable for 20 min] to form a purple coloured chromophore that has an absorption maxima at 554 nm. The method was validated as per ICH guidelines.
Results: Beer’s law was obeyed over the concentration range of 16-24 μg/mL for Cilnidipine. Limit of detection [LOD] and limit of quantification [LOQ] are 0.4365 and 1.3228 respectively. The average recovery was found to be 100.43% w/w.
Conclusion: The method developed is precise, linear and accurate. Satisfactory results were obtained from validation of the method. Hence the proposed method is suitable in quality control of estimation Cilnidipine bulk and pharmaceutical dosage forms.
Keywords: Cilnidipine, Visible spectrophotometry, N-(1-Naphthyl) ethylene diamine dihydrochloride, Validation, ICH guidelines.
Cilnidipine [CIL] is chemically, 1,4- Dihydro- 2,6- dimethyl- 4-(3-nitrophenyl)-3,5-pyridinecarboxylic acid 2-methoxyethyl(2E)-3-phenyl-propenyl ester. It is a dual blocker of L-type voltage-gated calcium channels in vascular smooth muscle and N-type calcium channels in sympathetic nerve terminals that supply blood vessels[1,2].
The literature survey revealed the lack of a visible spectrophotometric method for the estimation of CIL. However estimation of the drug as a single molecule and in combination with other drugs by UV spectrophotometric, HPLC and HPTLC was available[3-17].
The aim of present work was to develop an improved spectrophotometric method with greater precision and accuracy. The proposed method is mainly based on the reaction of Cilnidipine with N-(1-Naphthyl) ethylene diamine dihydrochloride (NED) reagent which gives purple color chromophore that has an absorption maximum at 554 nm. The structural formula for CIL is shown in Fig 1.
Fig. 1: Structure of Cilnidipine (C27H28N2O7)
MATERIALS AND METHODS
A Shimadzu UV/visible double beam spectrophotometer (model Lambda 25) with 1 cm matched quartz cells were used for all the spectral measurements.
Chemicals and Reagents
Pharmaceutical grade Cilnidipine was obtained as gift samples from Ideal Analytical and Research Institute, India. The pharmaceutical dosage form used in this study was Cilacar tablets (J. B. Chemical and Pharmaceuticals Ltd., Mumbai, India) labeled to contain 20 mg of Cilnidipine was procured from the market. Double distilled methanol was used. All other chemicals used were of Analar grade. All the aqueous solutions were prepared in double distilled water. 2 M hydrochloric acid, Sodium nitrite (0.1%w/v), Ammonium sulphamate (0.5%w/v), NED reagent (0.1%w/v) solutions were prepared in double distilled water.
Preparation of stock solution
The stock solution was prepared by dissolving 100 mg pure Cilnidipine in 25 mL of methanol in to 100 mL volumetric flasks and sonicated for 15 min. Then volume was made up to 100 mL with distilled water to give a concentration of 1 mg/mL.
Recommended procedure and calibration graph
Transfer increasing volumes of the filtrate, covering the range 16 – 24 mg/mL into a series of 50 mL volumetric flasks. Add 5 mL of 2M HCl and the mixtures are shaken. Then 5 mL of 0.1 %w/v sodium nitrite solution is added and the mixtures are allowed to stand for 2 minutes. Then 5 mL of 0.5 %w/v ammonium sulphamate solution is added and is allowed to stand for 2 minutes. After that 5 mL of 0.1 %w/v NED was added and the volumes are made up to the mark with methanol. After 10 minutes, the absorbance of the Purple chromogen was measured against a reagent blank, prepared in the same manner but without CIL at 554 nm.
Procedure for the tablet dosage form
Ten tablets (20 mg / tablet) were weighed and finely powdered. A portion of the powder equivalent to 100 mg of the drug was weighed in to a 100 mL volumetric flask and dissolved in 25 mL of methanol and was sonicated for 15 minutes. Then the volume was madwe upto the mark with distilled water. The solution was shaken well, filtered and an aliquot of the filtered drug solution was the treated as done in the recommended procedure.
The developed method was validated as per the International Conference on Harmonization (ICH) [18-19] guidelines with respect to linearity and range, precision, accuracy, limit of detection (LOD) and limit of quantization (LOQ). The values were analyzed by statistical methods.
The linearity of measurement was evaluated by analyzing different concentrations of the standard solution of CIL. Beer-Lambert’s concentration range was found to be 16-24 μg/mL. The overlay spectra and calibration graph are shown in Figure 2 and 3 respectively. The optical parameters are given in Table 1.
Fig. 2: Overlay spectrum of cilnidipine showing linearity
Fig. 3: Calibration curve of cilnidipine
Limit of detection (LOD) and limit of quantitation (LOQ)
The LOD and LOQ of CIL were determined by using standard deviation of the response and slope approach as defined in the International Conference on Harmonization (ICH) guidelines. The LOD and LOQ data presented in Table 1.
To ascertain the accuracy of proposed method, recovery studies were carried out by standard addition method at three different levels (80%, 100% and 120%). Percent recovery of CIL was found in the range of 98.3 – 102 %w/w (Table 2).
Table 1: Optical Characteristics and Other Parameters
|Regression equation||y = 0.0208X + 0.0084|
|Beer’s law limits (μg/mL)||16-24|
Table 2: Recovery Study Data.
|Preanalysed Quantity||Amount of pure drug added||Amount recovered (μg/mL)||% Recovery|
Precision is determined by using the same method used to assay the sample and repeated for a sufficient number of times to obtain statistically valid result. The experimental values were determined by Intra and interday. The values are validated by statistical analysis. The precision is then expressed as the relative standard deviation. % RSD was found to be 1.08665 and 0.89205 for intraday and Interday respectively.
RESULTS AND DISCUSSION
CIL is a new calcium channel antagonist used as antihypertensive agent. It showed maximum absorbance at 240 nm. Linearity was obeyed in concentration range of 3-18 μg/ml. As the values of % RSD of all precision study were within the acceptable limits (less than 2 %), the method provides good precision and reproducibility. The % RSD less than 2 indicated that the method was accurate. Results of the recovery study were found to be within the acceptance criteria linear, accurate, precise and reproducible.
A visible Spectrophotometric method has been developed for the determination of CIL in bulk and tablet formulation. The method was validated based on ICH analytical method validation guidelines. The method was found to be accurate, linear, precise and reproducible. Hence the method can be used for routine analysis of CIL in bulk and tablet formulation.
CONFLICT OF INTERESTS
The authors would like to thank the management of SRM College of Pharmacy, SRM University Chennai for providing the necessary infrastructural facilities to perform this study. We are highly grateful to Ideal analytical and research Institution, Puducherry India for providing gift sample of Camylofin dihydrochloride.