Metal complexation with the use of an auxiliary ligand is explored as an alternative to conventional protonation or deprotonation for analysis of a series of flavonoids by electrospray ionization mass spectrometry. Use of a neutral auxiliary ligand, 2,2'-bipyridine, results in formation of [MII(flavonoid - H)bpy]+, ternary complexes with intensities that are 2 orders of magnitude greater than the corresponding protonated flavonoids and up to 1.5 orders of magnitude greater than the deprotonated flavonoids, based on confirmation by collisionally activated dissociation patterns. The formation of ternary complexes with six divalent transition metals, Co2+, Ni2+, Cu2+, Zn2+, Mn2+, and Fe2+ were compared. Cu2+ resulted in the most intense complexes and simplest mass spectra, while Co2+ gave the second most intense spectra and also produced two key products that could be useful for a selected ion monitoring strategy. Complexation with iron(III) bromide is also investigated to explore the feasibility of using triply charged metals.

Collisionally activated dissociation is used for structural characterization of a series of flavonoid glycosides. Dissociation of transition metal/flavonoid binary complexes of the type [MII(L - H+)]+ and transition metal/2,2'-bipyridine/flavonoid ternary complexes of the type [MII(L - H+)bpy]+ give fragmentation patterns that are complementary and more diagnostic than those of the protonated, deprotonated, or sodium-cationized flavonoids. Analysis of fragmentation patterns of the [MII(L - H+)bpy]- complexes permits determination of the disaccharide as a rutinose or neohesperidose and the relative placement of the disaccharide (i.e., 3 vs. 7 positions).

The relative binding energies of a series of pyridyl ligand/metal complexes of the type [M(I)L(2)](+) and [M(II)L(3)](2+) are investigated by using energy-variable collisionally activated dissociation in a quadrupole ion trap mass spectrometer. The pyridyl ligands include 1,10-phenanthroline and various alkylated analogues, 2,2'-bipyridine, 4,4'-dimethyl-2,2'-bipyridine, and 2,2':6',2' '-terpyridine, and the metal ions include cobalt, nickel, copper, zinc, cadmium, calcium, magnesium, lithium, sodium, potassium, rubidium, and cesium. The effect of the ionic size and electronic nature of the metal ion and the polarizability and degree of preorganization of the pyridyl ligands on the threshold activation voltages, and thus the relative binding energies of the complexes, are evaluated. Correlations are found between the binding constants of [M(II)L(3)](2+) complexes in aqueous solution and the threshold activation voltages of the analogous gas-phase complexes determined by collisionally activated dissociation.

Sensitive and precise analytical methods are needed for flavonols, a subclass of flavonoids that has strong antioxidant activity. We report an improved method for identifying the predominant flavonols, quercetin and kaempferol, by collisionally activated dissociation (CAD) and quantifying them by high-performance liquid chromatography electrospray ionization mass spectrometry (HPLC-ESI-MS) in the selected ion monitoring mode. Practical applications of the method were demonstrated using several kale and biological samples. Two commercial kale samples were found to have 77 or 244 ppm quercetin and 235 or 347 ppm kaempferol (ppm = microg of quercetin/g of kale or microg of kaempferol/g of kale by fresh weight, 5-15% relative standard deviation). Blanching was found to reduce the flavonols to approximately 60% of the levels found in the unblanched kale. Isotopically labeled kale (cultivar Vates) grown in a greenhouse under an atmosphere of (13)CO(2) was found to have much lower flavonol levels. UV-A and UV-B supplementation during kale growth in the greenhouse was found to enhance both quercetin and kaempferol levels in Vates kale. The UV-B-supplemented kale not only produced more flavonols but the quercetin-to-kaempferol ratio was also higher than the UV-A-supplemented or the nonsupplemented kale. Recovery of flavonols from kale was approximately 60% based on spike and recovery trials with rutin, a glycoside of quercetin. Recovery of flavonols from biological samples spiked with rutin ranged from 96% for urine to 70% for plasma. Compared to UV detection, ESI-MS in the deprotonation mode provided lower detection limits, and both higher sensitivity and selectivity, in addition to structural characterization of the kale flavonols by CAD.