Method Development and Validation for the Determination of Caffeine: An Alkaloid from Coffea arabica by High-performance Liquid Chromatography Method

OBJECTIVE: The present study was investigated to develop and validate a reversed phase high performance liquid chromatography method for the determination of caffeine from bean material of Coffee arabica. MATERIALS AND METHODS: The separation was achieved on a reversed-phase C18 column using a mobile phase composed of water: methanol (50:50) at a flow rate of 1.0 mlmin-1. The detection was carried out on a UV detector at 272 nm. The developed method was validated according to the requirements for International Conference on Harmonisation (ICH) guidelines, which includes specificity, linearity, precision, accuracy, 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 peak area was less than 1% shows high precision of the method. The recovery rate for caffeine was within 98.78% - 101.28% 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 C. arabica at low concentrations. CONCLUSION: The developed HPLC method is a simple, rapid, precisely, accurately and widely accepted and it is recommended for efficient assays in routine work. SUMMARY: A simple, accurate, and sensitive high-performance liquid chromatography (HPLC) method for caffeine from Coffea arabica has been developed and validated. The developed HPLC method was validated for linearity, specificity, precision, recovery, limits of detection, and limits of quantification by the International Conference on Harmonization guidelines. The results revealed that the proposed method is highly reliable. This method could be successfully applied for routine quality work analysis. Abbreviation Used: C. arabica: Coffee arabica, ICH: International Conference on Harmonisation, % RSD: Percentage Relative Standard Deviation, R2: Correlation Coefficient, ppm: Parts per million, LOD: Limits of detection, LOQ: Limits of quantification, SD: Standard deviation, S: Slope, RP-HPLC: Reverse phase high performance liquid chromatography, v/v: Volume per volume. [Image: see text]