Plasma allantoin measurement by isocratic liquid chromatography with tandem mass spectrometry: method evaluation and application in oxidative stress biomonitoring.

Background: Allantoin in human plasma is a specific biomarker of oxidative stress. We describe a sensitive method to measure plasma allantoin using isocratic liquid chromatography and mass spectrometry (LC-MS/MS).

Methods: Direct injection of deproteinized plasma into the LC-MS/MS system was performed. The method was technically evaluated. Results on 200 healthy and 35 Type 2 diabetic Chinese subjects were compared.

Results: Dose-response of allantoin was linear to at least 21 pmol (20 μmol/l in plasma); LOD was 0.16 pmol; recovery 99.2-100.2% at 1-5 μmol/l; accuracy, 98.5-100.8%; within-day and between-day CVs (n=6), <4.0% (at 5.00-40 μmol/l) and <2.0% (at 1-5 μmol/l), respectively. Plasma allantoin in diabetic patients was ~8-fold higher than in healthy subjects; mean (SD): 8.82 (7.26) and 1.08 (0.86) μmol/l, respectively (p<0.0001). Allantoin was slightly higher in healthy men than in age- and BMI-matched women: 1.21 (0.99) μmol/l, n=88 compared to 0.97 (0.74) μmol/l, n=112; p<0.001. No association with age was seen. Gender difference was also seen in the diabetes patients: men, n=14, 11.57 (8.57) μmol/l; women, n=21, 6.99 (5.75) μmol/l, p<0.05.

Conclusions: Based on 95th percentiles of the healthy subjects, plasma allantoin of >2.2 μmol/l in women and >3.1 μmol/l in men indicates increased oxidative stress. Allantoin in diabetes subjects is clearly and markedly increased. The method will facilitate future studies of oxidative stress in human biomonitoring studies.