Rapid determination of methylxanthines in real samples by high-performance liquid chromatography using the new FastGradient narrow-bore monolithic column.

The present study reports one of the very first analytical applications of the new narrow-bore monolithic column, FastGradient Chromolith (50mm x 2.0mm i.d.). The three major methylxanthines (theobromine, theophylline and caffeine) were separated rapidly and determined simultaneously in various real samples. Based on the unique characteristics of this novel monolithic column the analytes were separated efficiently (R(s)>3.0) in less than 5min at a low flow rate of 0.5mLmin(-1) and using a low volume fraction of organic solvent (5% acetonitrile (ACN) in water) in the mobile phase. UV detection was carried out at 274nm. The separation was optimized in terms of mobile phase composition, flow rate and injection volume, while the method was validated for linearity, detection and quantitation limits, within and day-to-day precision, accuracy and ruggedness. Its applicability was demonstrated by analyzing a variety of real samples including beverages, soft drinks, herbal products and pharmaceuticals. Compared to a well-established monolithic (Performance Chromolith 100mm x 4.6mm i.d.) and a particulate reversed phase column (Hypersil ODS 5microm 150mm x 4.6mm i.d.), the narrow-bore FastGradient column offered satisfactory performance, faster analysis time and drastic reduction in the consumption of mobile phase and organic solvents.