Identification of isomeric flavonoid glucuronides in urine and plasma by metal complexation and LC‐ESI‐MS/MS

Abstract

Noncovalent complexes were used for structural determination and isomer differentiation of flavonoid glucuronides. Several flavonoid glucuronides including naringenin-7-O-glucuronide, synthesized here for the first time, were used as test compounds. Electrospray ionization quadrupole ion trap mass spectrometry with collision-induced dissociation (CID) was used to analyze complexes of the form [Co(II) (L-H) (Aux)]+ and [Co(II) (L-H) (Aux)2]+, in which L is the flavonoid glucuronide and Aux is a phenanthroline-based ligand. These complexes yielded characteristic fragmentation patterns that facilitated assignment of the substitution position of the glucuronides. The methods were adapted to liquid chromatography/tandem mass spectrometry (LC-MS/MS) with postcolumn cobalt complexation and were tested on extracts from biological fluids. The metabolites naringenin-7-O-glucuronide and naringenin-4'-O-glucuronide were detected in human urine following the consumption of grapefruit juice. Isomeric quercetin glucuronides were identified and differentiated after spiking rat plasma at the 1 microM level, proving that the new methods are effective at biologically relevant concentrations.

Regioselectivity of human UDP-glucuronosyl-transferase 1A1 in the synthesis of flavonoid glucuronides determined by metal complexation and tandem mass spectrometry

“…The site of conjugation is pinpointed based on interpretation of the MS/MS spectra of metal complexes of the type [Co(II) (FG Ϫ H) (4,7-dpphen) 2 ] ϩ . As discussedèinèanèearlierèreportè [31],ètheècharacteristic signs of 7-O-glucuronidation from this type of metal complex include significant losses of the glucuronic acid moiety (ϪGlcA) and of the auxiliary ligand (ϪAux), both individually and concurrently, as well as the loss of the flavonoid aglycon (ϪAgl), which is a diagnostic fragment for the 7-O-glucuronidation position. The metal complexes of B-ring-glucuronides typically display significant amounts of the ϪAux and Ϫ(Aux ϩ GlcA) fragment ions upon CID, but yield only minorèamountsèofètheèϪGlcAèfragmentèionè [31].èUpon CID, metal complexes of flavonol 3-O-glucuronides produce the Ϫ(Aux ϩ GlcA) fragment ion predominantlyè [31].è Supplementalè Figureè S1(whichè canè be found in the electronic version of this article) shows proposed structures for these general types of product ions.…”

“…The relative abundances of these ions are lower for the complex involving the flavonol (kaempferol) 7-O-glucuronide, and lowest for those complexes involving flavone (apigenin and acacetin) 7-O-glucuronides. The analogous complex involving baicalin (a flavone 7-O-glucuronide) was reported to show similarly low abundances of these fragment ions [31].…”

“…However, the -GlcA fragment ion appears to be an intrinsic part of the spectrum for the complexes of the quercetin and myricetin B-ring-glucuronides because there are no co-eluting isomers in these cases. A reasonable guideline is that the MS/MS of flavone 7-O-glucuronide complexes displays at least 15% relativeèabundanceèofètheèϪè GlcAèfragmentèionè (Figureè2aèandèc;èFigureè4a),èbutèthisèfragmentèionèis absent from the MS/MS of flavone B-ring-glucuronideècomplexesè(Figureè5a).èForèflavonols,ètheèsame product ion appears with ϳ50% relative abundance forètheè7-O-glucuronidesè(Figureè2d,èFigureè4cèandèe), but with ϳ10% relative abundance for B-ring-glucuronidesè(Figureè5cèandèe).èSinceèproductèionèdistribu-tionsèareèdependentèonècollisionèenergyè [31],èthese guidelines are only applicable to the conditions used in this set of experiments.…”

“…Most recently, we reported a metal complexation/ tandem mass spectrometry method for differentiating the conjugation positions of monoglucuronidated flavonols, flavones, and flavanones [31]. The metal complexation strategy entailed formation of complexes of the type [Co(II) (FG Ϫ H) (4,7-dpphen) 2 ] ϩ where (FG Ϫ H) is one molecule of deprotonated flavonoid glucuronide and (4,7-dpphen) 2 are two molecules of an auxiliary ligand, 4,7-diphenyl-1,10-phenanthroline.…”

Iron Chelating Activity of Gossypitrin Isolated from the Petals of Talipariti elatum Sw. (Fryxell) Malvaceae

“…This ability to chelate metals has been used to enhance the capabilities of MS; it has been used to assist the elucidation of flavonoid glucuronides (Davis et al, 2006;Davis et al, 2007) and various diglycosides (Pikulski et al, 2007). This is a result of the spectral changes observed when flavonoids are complexed with metals, giving rise to simpler yet more intense spectra (Satterfield & Brodbelt, 2000).…”