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.

Figures

Fig. 1

The pathway and structures of UA and its metabolites.

Fig. 2

Extracted SRM chromatograms of UA metabolites standards spiked in water. All of them were detected in ESI positive mode. (A) 6AU (6-aminouracil, 0.2 μmol/L, 4.13 min), (B) triuret (6.0 μmol/L, 5.37 min), (C) allantoin (6.0 μmol/L, 2.83 min), and (D) 13C9,15N-tyrosine (20.0 μmol/L, 5.29 min, ISTD).

Fig. 3

Chromatograms of the detected UA metabolites in urine sample from healthy volunteer. Urine samples were diluted 10-fold and filtered as described. (A) 6AU (RT: 4.14 min, S/N 83, 2.49 μmol/L), (B) triuret (not detected in urine from healthy, non-smokers), (C) allantoin (RT: 2.85 min, S/N 203, 3.10 μmol/L), and (D) 13C9,15 N-tyrosine (RT: 5.29 min, S/N 3659, IS). The value is absolute value.

Fig. 4

Chromatograms of triuret detected in urine from a heavy smoker and non-smoker. All urine was diluted 10-fold. (A) Triuret is present in the urine of a smoker, (B) ISTD in the urine of a smoker, (C) triuret in the urine of a non-smoker, (D) ISTD in urine from non-smoker. ISTD is 13C9,15 N-tyrosine.

Fig. 5

Triuret concentrations in the urine samples from smoker (N = 6, 0.34 ± 0.04 μg/mg creatinine) and non-smoker healthy volunteers (N = 21, 0.12 ± 0.10 μg/mg creatinine).