A highly sensitive assay for xanthine oxidoreductase activity using a combination of [13C2,15N2]xanthine and liquid chromatography/triple quadrupole mass spectrometry

In this study, we developed a highly sensitive assay for xanthine oxidoreductase (XOR) activity utilizing a combination of ¹³C₂,¹⁵N₂]xanthine and liquid chromatography (LC)/triple quadrupole mass spectrometry (TQMS). In this assay, the amount of ¹³C₂,¹⁵N₂]uric acid (UA) produced by XOR was determined by using LC/TQMS. For this assay, we synthesized ¹³C₂,¹⁵N₂]xanthine as a substrate, ¹³C₂,¹⁵N₂]UA as an analytical standard, and ¹³C₃,¹⁵N₃]UA as an internal standard. The ¹³C₂,¹⁵N₂]UA calibration curve obtained using LC/TQMS under the selected reaction monitoring mode was evaluated, and the results indicated good linearity (R² = 0.998, weighting of 1/x²) in the range of 20 to 4000 nM. As a model reaction of less active samples, the XOR activity of serial‐diluted mouse plasma was measured. Thereby, the XOR activity of the 1024‐fold‐diluted mouse plasma was 4.49 ± 0.44 pmol/100 μL/h (mean ± standard deviation, n = 3). This value is comparable to the predicted XOR activity value of healthy human plasma. Hence, this combination method may be used to obtain high‐sensitivity measurements required for XOR activity analysis on various organs or human plasma.