A simple, rapid, and robust ultrahigh-pressure liquid chromatography (UHPLC) method for the simultaneous determination of polyphenols, methylxanthines, sweeteners, and flavouring substances as well as some common preservatives has been developed using an automated method scouting and method optimization workflow. The most suitable mobile phase and stationary phase combination was identified in an overnight scouting run. These conditions were used to create a two-dimensional model using computer simulation software. Temperature and gradient time were varied to establish the best separation conditions. This approach resulted in a 1.5 min gradient method that could screen 17 compounds of interest.
However, a high cocoa and a high flavonoid content comes with an even higher amount of less desirable alkaloids, namely the stimulants caffeine and theobromine. In addition to the natural products found in cocoa beans, a large number of permitted additives can be added to chocolate products to alter the taste, texture, or expiry date, such as artificial or natural sweeteners, and flavouring compounds or preservatives. To monitor the composition and therefore the quality of cocoa in the food product, an analytical method needs to enable individual quantification of any of these potential ingredients.This article describes the development and optimization of a rapid ultrahigh-pressure liquid chromatography (UHPLC) screening test for the separation and quantification of polyphenols, methylxanthines, sweeteners, and flavouring substances as well as some common preservatives for the quality control of dark chocolate products.
Equipment and Chromatographic Methods: For UHPLC method scouting, a Shimadzu Nexera X2 Method Scouting System was used, consisting of two quaternary solvent pumps, autosampler, and column oven, including a six-column switching valve. The system was also equipped with a high resolution UV photo diode array (PDA) detector and an LCMS-8040 triple quadrupole mass spectrogmeter via an electrospray ionization (ESI) source.
Method scouting was performed in an overnight sequence using 5 min gradient runs at 40 °C. Five combinations of stationary phase and mobile phase were selected. The columns comprised a variety of bonded phases that exploit multiple mechanisms of separation and therefore provide alternative selectivity to common C18 phases. The aqueous and organic mobile phase combinations contained the same buffer salt/acid (AA/BA; AB/BB; and AC/BC).
The software predicted an optimum separation with a minimum resolution of the critical peak pair of 1.6 in a gradient run from 5% to 85% B in 1.5 min at a flow rate of 1.2 mL/min at 42 °C.
Sample Preparation: The chocolate sample was defatted and the compounds extracted into a sample solvent suitable for high performance liquid chromatography (HPLC) analysis.