Determination of Caffeine,Theobromine,and Theophylline in Standard Reference Material 2384,Baking Chocolate,Using Reversed-Phase Liquid Chromatography

摘要

A rapid and selective isocratic reversed-phase liquid chromatographic method has been developed at the National Institute of Standards and Technology to simultaneously measure caffeine,theobromine,and theophylline in a food-matrix standard reference material (SRM) 2384,Baking Chocolate.The method uses isocratic elution with a mobile phase composition (volume fractions) of 10% acetronitrile/90% water (pH adjusted to 2.5 using acetic acid) at a flow rate of 1.5 mL/min with ultraviolet absorbance detection (274 nm).Total elution time for these analytes is less than 15 min.Concentration levels of caffeine,theobromine,and theophylline were measured in single 1-g samples taken from each of eight bars of chocolate over an eight-day period.Samples were defatted with hexane,and /3-hydroxyethyltheophylline was added as the internal standard.The repeatability for the caffeine,theobromine,and theophylline measurements was 5.1,2.3,and 1.9%,respectively.The limit of quantitation for all analytes was <100 ng/mL.The measurements from this method were used in the value-assignment of caffeine,theobromine,and theophylline in SRM 2384.