Available online at www.ijntps.org ISSN: 2277 2782 INTERNATIONAL JOURNAL OF NOVEL TRENDS IN PHARMACEUTICAL SCIENCES Analytical Method Development and Validation of Caffeine in Tablet Dosage Form By Using RESEARCH ARTICLE S.Sethuraman 1, K.Radhakrishnan 2, T.Arul Solomon 2 1 Department of chemistry, SCSVMV University, Kanchipuram 2 Department of pharmaceutical, Jagans college of pharmacy, Nellore. Article Info Article history Received 25 Sep 2013 Revised 8 Oct 2013 Accepted 17 Oct 2013 Available online 30 Oct 2013 Keywords Caffeine, validation. UV-spectroscopy, Abstract Analytical method development and validation plays important role in the discovery, development and manufacture of pharmaceuticals. A simple, rapid and reproducibe UV- spectrophotometric method for the quantitative determination of caffeine in tablet formulation was developed and validated in the present work. The parameters lineartiy, specificity, precision, accuracy, robustness and ruggedness was studied according to ICH guidelines. The wavelength 270nm was selected for the estimation of drug using distilled water as a solvent. The drug obeyed Beer-lamber ts law over the concentration range 10-50µg/ml. The accuracy of the method was assessed by recovery studies and was found between 99.46-100.67%. The method was successfully applied for routine of this drug in formulations. INTRODUCTION Caffeine is a central nervous system and metabolic stimulant, and is used both recreationally and medically to reduce physical fatigue and to restore alertness when drowsiness occurs [1-3]. It produces increased wakefulness, faster and clearer flow of thought, increased focus, and better general body coordination. According to literature survey, it revealed that caffeine was quantitatively assayed in Tea, coffee and food beverages using liquid chromatography, UV-Visible spectroscopy How-ever no UV-Spectrophotometry method was proposed for the estimation of caffeine by using of water as a solvent in Tablet dosage forms [4-7]. In the present study to develop a simple, accurate and precise UV spectroscopic method for estimation of caffeine in tablet dosage form. The validation carried out as per ICH guidelines [8-10]. EXPERIMENTAL WORK Apparatus A Systronics double beam UV visible spectrophotometer model 2202, band width of 2nm wavelength accuracy ±0.5nm and two matched quartz cells with 1cm pathlength was used for all spectral measurements. Materials All the chemicals used were of analytical grade. A gift sample of caffeine obtained from Microlabs, Hosur, was used as working standard. The formulation of caffeine tablet was purchased from online shopping. Solubility test Solubility test for the drug Caffeine was performed by using various solvents. The solvents include Water, Methanol, Ethanol, Acetonitrile, Hydrochloric Acid (HCl), Sodium Hydroxide, Nitric acid, Sulphuric acid and Chloroform. DETERMINATION OF λ max Preparation of stock solution Standard stock solution of Caffeine was prepared by dissolving 100mg of Caffeine in 100ml of distilled water which gives 1000 µg/ml. Preparation of working solution From the above stock solution 10ml was transferred into 100ml volumetric flask and volume was made up to the mark with distilled water to make 100µg/ml. Then the sample was scanned with To whom correspondence should be addressed: S.Sethuraman Email: su_sethuraman@yahoo.com VOLUME 3 NUMBER 4 OCT 2013 82
UV-Vis Spectrophotometer in the range 200-400nm against distilled water as blank and the wavelength corresponding to maximum absorbance was noted which is its λ-max i.e. at 270nm. Fig 1. λ-max of Caffeine Preparation of calibration curve One ml of this 100µg/ml solution was further diluted and the volume was made up to 10ml by using method to produce 10µg/ml solution. 2ml, 3ml, 4ml and 5ml of 50µg/ml solution were diluted and the volume was made up to 10ml using methanol to produce 20µg/ml, 30µg/ml, 40µg/ml, 50µg/ml solutions respectively. Then the construction of calibration curve was done by taking the above prepared solutions of different concentration ranging from 10-50µg/ml. Then taking the absorbance calibration curve was plotted taking concentration on x-axis and absorbance on y-axis which showed a straight line. This straight line obeyed linearity in the concentration range of 10-50µg/ml. The correlation coefficient was found to be 0.999 Fig 2. Linearity curve of Caffeine Assay of Caffeine Tablet (200mg) A quantity of powder equivalent to 100mg of Caffeine was taken in a 100ml volumetric flask and it was dissolved and diluted up to the mark with distilled water. The resultant solution was ultrasonicated for 5 minutes. The solution was then filtered using Whatmann filter paper No. 40. From the filtrate, appropriate dilutions were made with distilled water to obtain the desired concentration (50 µg/ml). This solution was then analysed in UV and the result was indicated by % recovery given in table 4. Method validation Validation is a process of establishing documented evidence, which provides a high degree of assurance that a specific activity will consistently produce a desired result or product meeting its predetermined specifications and quality characteristics. The validation for UV method development was performed using parameters like Linearity, Accuracy, Precision, Robustness, and Ruggedness. Linearity Various aliquots were prepared from the secondary stock solution (100 µg/ml) ranging from 10-50 µg/ml. The samples were scanned in UV-Vis Spectrophotometer against distilled water as blank. It was found that the selected drug shows linearity between the ranges of 10-50 µg/ml.(table no.3) Accuracy Solutions were prepared in triplicate at levels 80%, 100% and 120% of test concentration using Caffeine working Standard as per the test method and taken absorbance of each solution in triplicate. The recovery results showed that the proposed method has an acceptable level of accuracy for Caffeine which is from 80% - 120% of test concentration is from 99.51 % - 100.01 %(Table 4). Precision Precision of the method was demonstrated by intraday and interday variation studies. In intraday variation study nine different solutions of same concentration 50µg/ml were analyzed three times in a day i.e. from morning, afternoon and evening and the absorbance is note. In the interday variation studies, solution of same concentration 50µg/ml were analyzed three times for the three VOLUME 3 NUMBER 4 OCT 2013 83
consecutive days and the absorbance result mean, standard deviation and %RSD was calculated and summarised results given in table 4. Specificity Got spectrum in the range of 200nm to 400nm for appropriate concentration of sample, Blank, and placebo. Fig 3. Spectrum for blank Fig 4. Spectrum for caffeine tablet excipients Fig 5. UV- Spectrum of standard Caffeine Robustness Robustness of the method was determined by carrying out the under different λ-max i.e. at 268nm and at 272nm. The respective absorbances of 50µg/ml were noted and the result was indicated as %RSD and given in table no.1 Ruggedness Ruggedness of the method was determined by carrying out the by different analyst and the respective absorbance of 50 µg/ml was noted. The result was indicated as %RSD and given in table no.2 RESULTS AND DISCUSSION The drug was analyzed at 270nm in distilled water using UV-Visible spectrophotometer. Optical characteristics such as Beer s law limits, intercept and slope has been calculated using regression equation, which has been presented in Table 3. Precision was determined by analyzing the drug at particular concentration for five times on the same day. Inter day precision was determined similarly, analyzing the samples daily, for three consecutive days. The results are summarised in Table 4. To ensure the accuracy method, recovery studies were performed by standard addition method at 80%,100% and 120% levels of drug concentration, to the pre-analyzed samples and percent recovery values were calculated. Recovery experiment indicated the absence of interferences from the commonly encountered pharmaceutical additives and excipients. The linearity studies were performed by plotting different concentration of standard solution against their respective absorbances. Caffeine were found to be linear in the concentration range of 10-50µg/ml. Correlation co-efficient value were found to be 0.999, calibration curve shows that it obeys Beer s law limit within the concentration range. The proposed method was found to be simple, accurate, precise, simple, sensitive, robust and cost effective. The results of the validation tests were found to be satisfactory and therefore this method can be applied successfully for the estimation of Caffeine in Tablet dosage form. VOLUME 3 NUMBER 4 OCT 2013 84
Uv- Spectroscopy Table 1. Robustness At 268nm At 272nm 50 0.535 Mean=0.5353 50 0.535 Mean=0.5348 50 0.535 50 0.536 50 0.536 SD=0.00103 50 0.535 SD=0.00116 50 0.537 50 0.534 50 0.535 %RSD=0.19 50 0.533 %RSD=0.21 50 0.534 50 0.536 Table 2. Ruggedness Analyst 1 Analyst 2 50 0.535 Mean=0.5355 50 0.534 Mean=0.5348 50 0.536 50 0.535 50 0.535 SD=0.000837 50 0.535 SD=0.000753 50 0.535 50 0.534 50 0.537 %RSD=0.15 50 0.536 %RSD=0.14 50 0.535 50 0.535 Table 3. Optical characteristics of Caffeine Beer s law limit(µg/ml) 10-50µg/ml Correlation coefficient 0.999 Regression equation(y*) 0.009x + 0.042 Slope 0.009 Intercept 0.042 Table 4. Summary of validation Parameter Result Linearity indicated by correlation coefficient 0.999 Precision indicated by % RSD 0.26% Accuracy indicated by % recovery 99.9044% Specificity No interferences of impurity Range 10µg-50µg/ml Linear regression equation 0.009x + 0.042 Robustness indicated by %RSD 0.19% Assay indicated by % recovery 99.89% REFERENCE 1. Wanyika HN, Gatebe EG, Gitu LM, Ngumba EK and Maritim CW. Determination of caffeine content of tea and instant coffee brands found in the Kenyan market., African Journal of Food Science, june 2010, 4(6), 353-358. VOLUME 3 NUMBER 4 OCT 2013 85
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