The applicability of comprehensive two-dimensional gas chromatography (GC x GC) for flavonoids analysis was investigated by separation and identification of flavonoids in standards, and a complex matrix natural sample. The modulation temperature was optimized to achieve the best separation and signal enhancement. The separation pattern of trimethylsilyl (TMS) derivatives of flavonoids was compared on two complementary column sets. Whilst the BPX5/BPX50 (NP/P) column set offers better overall separation, BPX50/BPX5 (P/NP) provides better peak shape and sensitivity. Comparison of the identification power of GC x GC-TOFMS against both the NIST05 MS library and a laboratory (created in-house) TOFMS library was carried out on a flavonoid mixture. The basic retention index information on high-performance capillary columns with a non-polar stationary phase was established and database of mass spectra of trimethylsilyl derivatives of flavonoids was compiled. TOFMS coupled to GC x GC enabled satisfactory identification of flavonoids in complex matrix samples at their LOD over a range of 0.5-10 mu g/mL.. Detection of all compounds was based on full-scan mass spectra and for each compound a characteristic ion was chosen for further quantification. This study shows that GC x GC-TOFMS yields high specificity for flavonoids derived from real natural samples, dark chocolate, propolis, and chrysanthemum. (C) 2010 Elsevier B.V. All rights reserved.