Assay of serum allantoin in humans by gas chromatography-mass spectrometry

Background: The small amount of allantoin present in human serum results from free radical (FR) action on urate and may provide a stable marker of free radical activity in vivo. We describe a gas chromatography-mass spectrometry (GC-MS) assay for serum allantoin and report a reference range in healthy individuals. Methods: Fasting blood samples were obtained from 134 healthy middle-aged volunteers (56 men, mean age 55, range 45-72; 78 women, mean age 55, range 50-72) Allantoin was assayed using N-15(2) allantoin as an internal standard. After isolation from aqueous standards or serum by extraction onto an anion exchange column (AG-MP1), allantoin was derivatised with N-methyl-N-(tert-butyldimethylsilyl) trifluoroacetamide (MTBSTFA). Derivatives were injected onto an HP-1 column and analysed using a Mass Selective Detector with Single Ion Monitoring at 398 and 400 m/z. Results: The distribution of serum allantoin concentrations in men and women was non-Gaussian and log transformation was used for the analysis of data. Women (10.8 +/- 1.7 mumol/l (mean +/- S.D.)) had significantly lower serum allantoin levels than men (13.4 +/- 1.6 mumol/l, p = 0.015). Reference ranges (95% CI) for middle-aged healthy subjects were 7.4-46.8 mumol/l (men) and 3.7-31.2 mumol/l (women). Conclusion: Gas chromatography-mass spectrometry provides a reliable and accurate method for the determination of serum allantoin. (C) 2002 Elsevier Science B.V. All rights reserved.