Caffeine content of tea extracts and formulations may conveniently be assayed by a number of titrimetric, spectrophotometric, gravimetric and chromatographic methods. HPLC is the method of choice as it is subject to fewer interferences than other methods.

A. Method I (Spectrophotometric assay)

1. Sample preparation: 3 g tea sample is taken in a 250 ml beaker and 20 ml boiling purified water is added. It is stirred for 1 min on a magnetic stirrer (500 rpm) and allowed to cool to room temperature.
2. Standard preparation: A 1,000 ppm stock standard of caffeine is prepared by dissolving 198.2 mg of caffeine in 200 ml purified water. Working standards are prepared by pipetting 25, 12.5, 10, 7.5, 5 and 2.5 ml aliquots of the stock standard solution into separate 50 ml volumetric flasks and diluting to volume with purified water.
3. Absorbance of the solutions is measured at 260 nm against purified water as blank. Caffeine content is estimated from standard curve plotted from the absorbance values of the standard caffeine samples.

The method could be modified by extracting the tea infusion with dichloromethane. The organic layer is separated and its absorbance measured at 276 nm against dichloromethane blank. Caffeine content is determined from a similar standard curve of a standard solution of caffeine in dichloromethane.