Although the use of comprehensive two-dimensional liquid chromatography (LCxLC) as a powerful separation technique is continuously increasing, its employment in quantification experiments is rather limited. The present research is focused on the quantification of a series of standards, as well as real-world sample compounds, by using dedicated laboratory-constructed LCxLC software, developed through a novel approach. Moreover, the difficulties encountered during software operation, in various elution conditions, are described and discussed. The results attained were compared with those observed in conventional LC, and no statistically significant differences were observed in the determination of aurapten in grapefruit oil. However, a loss in sensitivity was observed when using LCxLC (limit of detection = 0.10 ppm) compared to conventional LC (limit of detection = 0.05 ppm) as a consequence of the sample dilution in comprehensive two-dimensional liquid chromatography.