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 [C-13(2),N-15(2)]xanthine and liquid chromatography (LC)/triple quadrupole mass spectrometry (TQMS). In this assay, the amount of [C-13(2),N-15(2)]uric acid (UA) produced by XOR was determined by using LC/TQMS. For this assay, we synthesized [C-13(2),N-15(2)]xanthine as a substrate, [C-13(2),N-15(2)]UA as an analytical standard, and [C-13(3),N-15(3)]UA as an internal standard. The [C-13(2),N-15(2)]UA calibration curve obtained using LC/TQMS under the selected reaction monitoring mode was evaluated, and the results indicated good linearity (R-2=0.998, weighting of 1/x(2)) in the range of 20 to 4000nM. 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 +/- 0.44pmol/100L/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.