Optimization and validation of HPLC–DAD method for simultaneous analysis of sweeteners, preservatives, and caffeine in sugar-free beverages

In this study, the optimization and performance evaluation of a high-performance liquid chromatography with diode array detection (HPLC–DAD) method for the simultaneous determination of four sweeteners (acesulfame-potassium, saccharin, aspartame, and rebaudioside A), two preservatives (sodium benzoate and potassium sorbate), and caffeine in sugar-free drinks are presented. The separation was carried out using a gradient elution of acetonitrile and phosphate buffer (12.5 mM, pH = 3.3) on a reversed-phase column. Under the optimized conditions, the method allowed for the specific and selective simultaneous separation of all target analytes in less than 9 min. The developed HPLC–DAD method was validated and demonstrated excellent linearity (all analytical curves showed R 2 ≥ 0.9995), satisfactory accuracy (recovery values ranging between 94.1 and 99.2% in real samples), and repeatability (intra- and inter-day relative standard deviations were ≤ 2.49%). Overall, 69 products available in the Hungarian market were successfully tested with the applied method.