title: Simultaneous determination of uric acid metabolites allantoin, 6-aminouracil, and triuret in human urine using liquid chromatography-mass spectrometry.
abstract: Uric acid (UA) can be directly converted to allantoin enzymatically by uricase in most mammals except humans or by reaction with superoxide. UA can react directly with nitric oxide to generate 6-aminouracil and with peroxynitrite to yield triuret; both of these metabolites have been identified in biological samples. We now report a validated high-performance liquid chromatography and tandem mass spectrometry method for the determination of these urinary UA metabolites. Urine samples were diluted 10-fold, filtered and directly injected onto HPLC for LC-MS/MS analysis. The urinary metabolites of UA were separated using gradient HPLC. Identification and quantification of UA urinary metabolites was performed with electrospray in positive ion mode by selected-reaction monitoring (SRM). Correlation coefficients were 0.991-0.999 from the calibration curve. The intra- and inter-day precision (R.S.D., %) of the metabolites ranged from 0.5% to 13.4% and 2.5-12.2%, respectively. In normal individuals (n=21), urinary allantoin, 6-aminouracil and triuret, were 15.30 (+/-8.96), 0.22 (+/-0.12), and 0.12 (+/-0.10) microg/mg of urinary creatinine (mean (+/-S.D.)), respectively. The new method was used to show that smoking, which can induce oxidative stress, is associated with elevated triuret levels in urine. Thus, the method may be helpful in identifying pathways of oxidative stress in biological samples.
authors.affiliation: Division of Nephrology and Hypertension, Department of Medicine, University of Florida, Gainesville, FL 32610-0226, USA. kyung.kim@medicine.ufl.edu
authors.forename: Kyung Mee
authors.initials: KM
authors.lastname: Kim
authors.forename: George N
authors.initials: GN
authors.lastname: Henderson
authors.forename: Reginald F
authors.initials: RF
authors.lastname: Frye
authors.forename: Cheryl D
authors.initials: CD
authors.lastname: Galloway
authors.forename: Nancy J
authors.initials: NJ
authors.lastname: Brown
authors.forename: Mark S
authors.initials: MS
authors.lastname: Segal
authors.forename: Witcha
authors.initials: W
authors.lastname: Imaram
authors.forename: Alexander
authors.initials: A
authors.lastname: Angerhofer
authors.forename: Richard J
authors.initials: RJ
authors.lastname: Johnson
mesh_terms: D000481:Allantoin / Q000652:urine*; D002138:Calibration; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D006801:Humans; D012015:Reference Standards; D015203:Reproducibility of Results; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D014498:Uracil / Q000031:analogs & derivatives* / Q000652:urine; D014508:Urea / Q000031:analogs & derivatives* / Q000652:urine
chemical_list: D000481:Allantoin; D014498:Uracil; C017781:triuret; C013400:6-aminouracil; D014508:Urea