Determination of flavonoids and stilbenes in red wine and related biological products by HPLC and HPLC-ESI-MS-MS

To investigate probable health benefits of flavonoids and stilbenes in red wine a new reversed-phase (RP) high-performance liquid-chromatographic (HPL C) method with enhanced separation efficiency and improved selectivity, sen sitivity, and speed has been established for determination of the flavonoid s quercetin, myricetin and kaempferol and the stilbenes cis- and trans-resv eratrol, in a single run. UV-absorbance, fluorescence (FLD), and mass-spect rometric (MS) detection were also evaluated. UV-absorbance detection at 320 nm for stilbenes and 377 nm for flavonoids enables their determination up to the nanogram range with a linearity of R-2 > 0.9999 (linear range 50 ng mL(-1) -50 mug mL(-1)). Calculated values of average recoveries were betwee n 95 and 105% for all analytes. For resveratrol, fluorescence detection was highly selective and twice as sensitive as UV detection, and linearity was satisfactory (R-2 > 0.9996; linear range see UV detection). For the detect ion of the hydrophilic glycosidic compounds piceid and rutin, which are coe luted with other hydrophilic ingredients, the validated RP HPLC system was coupled to a quadrupole ion-trap mass-spectrometer (MS) via an electrospray interface (ESI) with 25% ammonia solution as sheath liquid. MS detection w as, highly linear (R-2 > 0.9878; linear range 50 ng mL(-1) -50 pg mL(-1)) f or all investigated analytes and the limits of detection were in the low na nogram range. Compared with UV detection MS detection resulted in a 200% in crease in signal intensity for myricetin and 400% increases for quercetin a nd kaempferol, but equal signal intensity for resveratrol. Calculated value s of average recoveries were 102% for myricetin and 79% for piceid. Collisi on induced dissociation (CID) was also used to obtain characteristic fragme ntation fingerprints to facilitate qualitative and quantitative analysis ev en in complex matrices. Finally, this hyphenated HPLC-ESI-MS method was hig hly suitable and an essential improvement compared with UV- and fluorescenc e detection.