A rapid and selective isocratic reversed-phase liquid chromatographic method has
been developedat the National Institute of Standards and Technology to simultaneously measure caffeine,theo
bromine, and theophylline in a food-matrix standard reference material (SRM) 2384, BakingChocolate. The method uses isocratic elution with a mo
bile phase composition (volume fractions) of10% acetronitrile/90% water (pH adjusted to 2.5 using acetic acid) at a flow rate of 1.5 mL/min withultraviolet a
bsor
bance detection (274 nm). Total elution time for these analytes is less than 15 min.Concentration levels of caffeine, theo
bromine, and theophylline were measured in single 1-g samplestaken from each of eight
bars of chocolate over an eight-day period. Samples were defatted withhexane, and
beta2.gif" BORDER=0 ALIGN="middle">-hydroxyethyltheophylline was added as the internal standard. The repeata
bility forthe caffeine, theo
bromine, 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 wereused in the value-assignment of caffeine, theo
bromine, and theophylline in SRM 2384.Keywords: Standard reference material; caffeine; theo
bromine; theophylline; chocolate; liquid chromatography; reversed-phase