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An Innovative Approach to the Preparation of Plasma Samples for UHPLC-MS Analysis.

A new sample processing method for analyzing flavonol metabolites in plasma using enzymatic proteolysis was developed and validated. Four endopeptidases were examined regarding their influence on the analyte recovery of quercetin-3- O-glucuronide (Q3GlcA). Methanol was added to inactivate and precipitate the enzymes, and samples were concentrated via evaporation prior to UHPLC-MS analysis. Quercetin-3- O-rutinoside (Q3Rut) was used as an internal standard. The selectivity and accuracy of the established UHPLC-ESI-MS n method showed a coefficient of variation (CV) of the repeatability of the measuring instrument of 1.7% for Q3GlcA. The average recovery of Q3GlcA was approximately 67% with an interday method precision of 24% and r = 46.9 as its repeatability. Therefore, enzymatic proteolysis has proven to be a suitable alternative to the methods previously described in the literature, such as solid-phase extraction (SPE). Still, the method has only been validated for Q3GlcA, but its applicability to other substance classes seems possible.