Detection of allantoin in clinical samples using hydrophilic liquid chromatography with stable isotope dilution negative ion tandem mass spectrometry.
Allantoin is the major oxidation product of urate in humans and is a potential biomarker of oxidative stress. Several methods are used to measure allantoin in biological samples but they have inherent issues that can include lack of specificity and sensitivity, difficulty in sample preparation, or artefactual generation of allantoin. We have developed a method for measuring allantoin using hydrophilic liquid chromatography with stable isotope dilution tandem mass spectrometry (HILIC-MS/MS). It was validated for measuring allantoin in plasma, synovial fluid and urine from human subjects. The limit of quantification was determined to be 10 fmol and the assay displayed excellent linearity for the wide range of concentrations found in clinical samples. Relative standard deviations were <5% for between-day and <7% for within-day variation. Accuracy was between 100% and 104%. Concentrations of allantoin in plasma of healthy controls (2.0 μM; interquartile range 1.4-3.6 μM, n=35) was significantly lower (p<0.001) than that in plasma from patients with rheumatoid arthritis (3.7 μM; IQR 3.0-5.6 μM, n=43) and in synovial fluid of patients with gout (3.3 μM; IQR 2.8-5.8 μM, n=10). This newer HILIC-MS/MS method is a simple and highly sensitive assay for detection of allantoin. It can be used to assess the level of oxidative stress in human pathologies.