One‐Electron Oxidation and Sensitivity of Uric Acid in On‐Line Electrochemistry and in Electrospray Ionization Mass Spectrometry

The sensitivity of detection of uric acid (H2U) in positive ion mode electrospray ionization mass spectrometry (ESI MS) was enhanced by uric acid oxidation during electrospray ionization. With a carrier solution of pH 6.3>pKa1=5.4 of H2U, protonated unoxidized uric acid [H2U+H]+(m/z169) was detected together with the protonated uric acid dimer [2H2U+H]+(m/z337). The dimer likely forms by 1e−oxidation of urate (HU−) followed by rapid radical dimerization. A covalent structure of the dimer was verified by H/D exchange experiments. Efficiency of 2e−, 2H+oxidation of uric acid is low during ESI in pH 6.3 carrier solution and improves when a low on‐line electrochemical cell voltage is floated on the high voltage of the ES in on‐line electrochemistry ESI MS (EC/ESI MS). The intensity of the uric acid dimer decreases with an increase in the low applied voltage. In a carrier solution with 0.1 M KOH, pH 12.7>pKa2=9.8 of H2U, allantoin (Allnt) (MW 158.04), the final 2e−, 2H+oxidation product of uric acid, was detected as a potassium complex [K(Allnt)+K]+(m/z235) and the [2H2U+H]+dimer was not detected. In direct ESI MS analysis of 1000‐fold diluted urine [NaHU+H]+(pKspNaHU=4.6) was detected in 40/60 (vol%) water/methanol, 1 mM NH4Ac, pH ca. 6.3 carrier solution. A new configuration of the ESI MS instrument with a cone‐shaped capillary inlet significantly enhanced sensitivity in ESI and EC/ESI MS measurements of uric acid.