A sensitive and specific liquid chromatography-tandem mass spectrometry method for the determination of intracellular and extracellular uric acid

Abstract

Uric acid (UA) is known to be a major biological antioxidant in plasma. However, there is a strong correlation between UA levels and cardiovascular risk. Recent studies suggest that in the intracellular environment, UA can become a prooxidant that causes endothelial dysfunction. For conducting detailed studies of UA's role in human pathogenesis, there is a critical need for a sensitive and specific method for the determination of intracellular UA levels. We therefore developed a simple, sensitive method for determination of trace amounts of intracellular UA, as well as comparatively large amounts of UA in plasma and urine (for the determination of extracellular concentrations of UA), based on liquid chromatography and tandem mass spectrometry (LC-MS/MS). UA was separated from interferences by HPLC and quantified by mass spectrometry in the negative ESI mode using single reaction monitoring (SRM). For the identification and quantification of UA, the parent ions selected were m/z 167.0, which corresponds to the urate anion, and m/z 169.0, which corresponds to the 1,3-(15)N(2)-UA anion. 1,3-(15)N(2)-UA is used as an internal standard to ensure accuracy of the measurement. After precipitation of proteins with 10% TCA solution, UA was subjected to LC-MS/MS analysis. The correlation coefficient was 0.9998-1.0000 based on the calibration curve. The intra- and inter-day precision (C.V. %) ranged from 0.01 to 3.07 and 0.01 to 3.68 for in vivo and in vitro systems, respectively. Recovery tests of added standards have been successfully performed and the values ranged from 90.10 to 103.59% and 98.74 to 106.12% for in vivo and in vitro analyses, respectively. This study demonstrates that intracellular levels of UA can be measured using LC-MS/MS with isotope labeled UA as an internal standard.

Figure 1

SRM chromatograms of UA and 1,3-¹⁵N₂-UA standards. All of them were recorded at the collision energy of 25V and using ESI interface in the negative ion mode. (A); UA (1.00 mg/dl, up panel m/z 96.0: down panel is m/z 124.0), (B); 1,3-¹⁵N₂-UA (4.51 mg/dl, up panel m/z 97.0: down panel is m/z 125.0)

Figure 2

MS/MS spectra of UA and 1,3-¹⁵N₂-UA standards determined using various collision energies. All of them were performed in the negative mode using ESI interface. The major fragmentation ions of UA were m/z 124.0, 96.0, and 69.0 from molecular ion m/z 167.0. A: collision energy 15V for UA, B; collision energy 25V for UA, C; collision energy 30V for UA, and D; collision energy 25V for 1,3-¹⁵N₂-UA (fragmentation ions; m/z 125.0, 97.0, and 70.0 from molecular ion m/z 169.0)

Figure 3

SRM TIC chromatograms of UA in normal plasma (A: 4.73 mg/dl), urine (B: 15.75 mg/dl), and HUVEC cell lysate (C: 0.05 mg/dl). All of them were applied the collision energy of 25V and used ESI interface.

Figure 4

Time-dependent production of UA in kidney tubular cells after treatment with 5 mM fructose.