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Method Development and Validation for the Determination of Purine Alkaloid Caffeine from Camellia sinensis by RP-HPLC Method

Objective: This study was done with the objective of developing a validated method for routine analysis of caffeine content in tea by reversed phase high performance liquid chromatography method. Caffeine a purine alkaloid a xanthine derivative obtained from tea leaves Camellia sinensis. Methods: Caffeine crystals were isolated from tea leaves separated through a reversed-phase C18 Chromatopak (250 mm X 4.6 X 5 micron) column using a mobile phase composed of water: Acetonitrile (60:40) at a flow rate of 1.0 ml/min. The peak response time for caffeine was observed at 1.275 minutes using UV detector set at 273 nm. The developed method was validated according to the International Conference on Harmonisation (ICH) guidelines, which includes specificity, linearity, precision, accuracy, robustness, limit of detection and limit of quantitation. Results: The developed method validates good linearity with excellent correlation coefficient (R2>0.999). In repeatability and intermediate precision, the percentage relative standard deviation (% RSD) of results was less than 1% shows high precision of the method. The recovery rate for caffeine was within 97.4-98.6% indicates high accuracy of the method. The low limit of detection and limit of quantitation of caffeine enable the detection and quantitation of caffeine from Camellia sinensis at low concentrations. Conclusion: The developed RP-HPLC method is a simple, rapid, precise, accurate can be widely accepted and can be recommended for efficient assays. The run time was 30 mins the Rt was 1.275, the run time can be decreased to less than 10 mins, the mobile phase and run time can be saved.


Sivagami B, Chandrasekar R, Mohammad Ali S, Vamshi Krishna R, Mounika B, Deepa P, Divya P and Lawrence R

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