DEVELOPMENT OF NEW ANALYTICAL METHODS FOR THE QUANTIFICATION OF CAFFEINE, ORGANIC AND CHLOROGENIC ACIDS FROM ESPRESSO COFFEE USING SPE AND HPLC-DAD

Introduction Coffee (Coffea sp.) was first cultivated between the fifth and eighth centuries. The coffee beverage consists of over 1000 volatile and non-volatile compounds that are responsible for the taste and aroma 1. Of these, caffeine (1,3,7-trimethylxanthine) is the most studied compound, responsible for the stimulant effect on the central nervous system 2. Among the compounds that have the greatest influence on the taste and aroma of coffee, carboxylic acids, specific organic acids and chlorogenic acids were found. There are nearly 38 different organic acids (OAs)3. OAs are important because their properties allow us to define the type of acidity perceived in the beverage 4. Acids account for a large proportion of the total mass, up to 11% in green beans and 6% in roasted beans 3. In literature, different concentration ranges of organic acids were found in the coffee matrix, analyzed by several sample clean-up procedures and many chromatographic conditions. The term chlorogenic acids (CGA) encompasses many types of quinic and caffeic acids esters (combining quinic and caffeic acids through an oxygen atom), and as such, the structures of CGAs can become quite complex and large. During roasting, CGAs convert to CGA lactones, which contribute to the bitterness of roasted coffee. For this reason, after developing the sample preparation and the ideal chromatographic conditions, it was decided to apply these two new and fast analytical methods to analyze the different compositions of the nine organic acids, four chlorogenic acids, two phenolic compounds and caffeine in three different espresso coffees (EC) made from the same coffee treated with three roasting methods (light, medium and dark) 5, with the main objective of determining how the different roasting can affect the coffee composition Materials and Methods For the EC samples, were used three different roasting grades (light, medium and dark) of the same coffee variety 100% Arabica (Kenya Kaliluni) supplied by Perfero Coffee (Altidona, Italy). The EC samples were prepared using VA388 Black Eagle Maverick machine (Simonelli Group, Victoria Arduino). Before filtration and HPLC-DAD analysis, several methods were tried to purify the samples for OAs analysis, finally the anion exchange solid phase extraction were selected. Then, a new HPLC-DAD was validated for the analysis of OAs, for this was used an analytical column Luna Omega Polar C18 (250 mm × 4.6 mm, 3 μm) and elution was performed in isocratic mode using a potassium dihydrogen phosphate buffer at a pH of 2.5 at a flow rate of 0.8 mL/min. While the new method for analysis of chlorogenic and phenolic acids and caffeine used a Gemini C18 (250 mm × 3.0 mm, 5 μm) and elution was performed in gradient mode using water (A) and methanol (B), both with 0.1% formic acid as the mobile phase. Conclusions This research allowed further elucidation of the content of carboxylic acid in the samples of EC by developing innovative SPE purification procedures and HPLC methods. The efficiency of the developed analytical method and the affordability of the equipment used could simplify the determination of OAs, CGAs, phenolic acid, and caffeine in the complex matrix of coffee. With these methods, 9 OAs, 4 CGAs, 2 phenolic acids and caffeine were clearly and accurately detected in an Arabica sample EC. Finally, the caffeine content is relatively stable to heat. While the high concentration of chlorogenic acids decreases due to roasting, and the total organic acid content increases with medium and dark roasting due to the formation of acetic and lactic acids, but each acid shows a different trend based on the degradation or activation of formation processes. One of the most important innovations of the study is the short purification procedure using anion exchange solid phase extraction followed by direct HPLC-DAD analysis for the OAs. Therefore, this method can be considered as a reference method for the determination of carboxylic acids in coffee.