Assessment of the inaccuracy associated with the quantification of phenolic compounds using UHPLC-DAD and HPLC-MS/MS

The growing emphasis on health-conscious dietary practices has stimulated extensive research of phenolic and antioxidant compounds, particularly within horticultural food matrices. Their individual quantification, following separation and identification is commonly performed by (ultra-)high-performance liquid chromatography -(U)HPLC- coupled with UV-vis absorption or mass spectrometry. Given the extensive chemical diversity and the economic cost of high-purity standards, the use of equivalent standards to quantify multiple analytes with similar structures is a widespread practice. Despite the evident inaccuracy this approximation may introduce, it is scarcely elucidated in scientific literature. To clarify the magnitude of this error, external standard calibration was performed for 6 phenolic acids and 8 flavonoids using 2 liquid chromatographs, a UHPLC coupled with a diode array detector (DAD) and an HPLC with tandem mass spectrometry (HPLC-MS/MS). When calculations were expressed in terms of molarity, lower discrepancies were obtained by accounting for the molecular weight difference. The UHPLC-DAD analyses exhibited a lower degree of inaccuracy, particularly for flavonoids, no differences were observed when using eriocitrin, rutin, or luteolin to quantify a 16.8 µM eriocitrin solution, with slight variations for all. In contrast, HPLC-MS/MS showed different concentration values across all standards, except between naringenin and diosmin, and luteolin and rutin, which could be considered mutually equivalent. This study provides key insights into the relation between structural diversity and signal using UV-Vis absorption and mass spectrometry representing a starting point for future research focused on the accurate individual quantification of food compounds within their wide diversity.