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 at an undergraduate level before reaching industry.
This project will address the development of a method to train undergraduate students as well as instructors how to apply LC-MS/MS to various applications. The goal of the project is to teach students how to take advantage of the low detection limit when needed and to use the less sensitive methods with samples containing higher amounts of analytes. The power of the LC-MS/MS is that it can be used to detect several ranges of concentration on a single instrument with compounds exhibiting a broad range of polarities, essentially enabling a larger dynamic range. One of the major challenges is setting the activity up in such a way that students gain meaningful understanding of the instrument and the broad range of its capabilities while being very mindful of the time constraint and skill level of the students.
The two compounds that are the focus of this study are caffeine and theobromine. These are most commonly known as the stimulants in coffee and chocolate, respectively. These natural products are ideal for demonstrating the power of this instrument because they differ by a single methyl making the compound very difficult to separate, but easy to distinguish using mass spectrometry. A mixture of these compounds were analyzed using Scan, SIM and MRM methods to create calibration curves. The amount of caffeine and theobromine in each sample of dissolved chocolate was then determined by the students. The analyses ware performed on an Agilent Technologies 6410 Triple Quad LC/MS, using an Agilent Technologies 1260 Infinity LC.
A single pump gradient was established and used for all three of the analysis modes.
Linearity of the calibration curves were established between 0.1-0.7mg/mL in Scan mode, and below 0.1-0.7μg/mL using MRM mode, with the overall detection limit at just over 1 pg/mL. Students were able to operate instrument and use both qualitative and quantitative analysis programs.
There are several factors that make the LC-MS/MS a unique and powerful piece of lab equipment. It is rare in analytical instruments that clean peak resolution is not a necessity, but this is the case using tandem mass spectrometry as the instrument can be set to recognize individual compounds. As both caffeine and theobromine are frequently found at varying ratios in the same products or biological samples it is important to be able to look at these compounds together. This experiment effectively demonstrates to students the advantages of this instrument and increased their knowledge base moving into the workforce.