Analysis of Caffeine and Theobromine in Cocoa and Beer; LCMS Method Development for Undergraduate Lab Curriculum

Most modern research labs utilize liquid chromatography-tandem mass spectrometry, or LC-MS/MS, to analyze mixtures in both qualitative and quantitative capacities. This makes it imperative to teach students how to operate and understand these instruments even at an undergraduate level. The final goal of this project was to develop a method to use in an academic setting to demonstrate the versatility of LC-MS/MS. The protocol takes advantage of the low detection limit when needed, while using less sensitive methods with samples containing higher amounts of analytes. There are multiple methods used to run samples including Scan for broad range data at high concentrations and multiple reaction monitoring (MRM) for highly specific low concentration analysis. The dynamic range of the instrument was shown from 1 pg/mL to 1 mg/mL when run in the various available modes. The two compounds utilized in this study to demonstrate the unique abilities of this instrument are caffeine and theobromine. These are most commonly known as the stimulants in coffee and cocoa respectively. These natural products are ideal for exhibiting the power of this instrument because they are homologous compounds that differ by a single methyl group and have very similar polarity and spectroscopic characteristics. This makes it difficult to achieve rapid baseline separation using many instruments commonly found in a chemistry laboratory, but they can be individually integrated even without baseline separation using the MRM method. The high matrix nature of the food samples was overcome with the specificity of the method without extensive sample preparation. A rinse method that cycled between high and low organic mobile phase was shown to effectively elute residual compounds from the column. A lab protocol and instrumental instruction set were established for the Agilent 6410 triple quad system.